Innate-like T cells, namely natural killer T(NKT) and γδ T cells, play critical roles in linking innate and adaptive immune responses through rapid production of cytokines. Prominent among these cytokines is interle...Innate-like T cells, namely natural killer T(NKT) and γδ T cells, play critical roles in linking innate and adaptive immune responses through rapid production of cytokines. Prominent among these cytokines is interleukin-17(IL-17), which is a potent proinflammatory cytokine that plays a critical role in host defense against fungi and extracellular bacteria. However, excessive IL-17-production promotes autoimmune diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. IL-17 has also been implicated in regulating body fat, which is highly relevant given rises in obesity and type 2 diabetes. NKT cells, γδ T cells and mucosal-associated invariant T cells(MAIT) are the major sources of IL-17 involved in protection of mucosal surfaces from opportunistic infections and causing autoimmunity when become dysregulated. Given the pathogenic effects of IL-17, efforts have been directed towards understanding mechanisms that guard against IL-17 overproduction. One novel potent mechanism is mediated by the heparan sulfate proteoglycan, syndecan-1(sdc1), which is selectively expressed by IL-17-producing subsets of NKT and γδ T cells. This unexpected role for sdc1 is uncovered by analysis of NKT and γδ T cells in sdc1-deficient mice. In this mini-review, we discuss selective expression of sdc1 by these innate T cells and consequences of its absence on IL-17 homeostasis and pathological implications.展开更多
· AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptid...· AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptides1169 to 1191 of the interphotoreceptor binding protein(IRBP). Rapamycin(0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4 +CD25 + regulatory T cells(Tregs)from rat spleen were detected by flow cytometry.·RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro.Rapamycin also significantly increased TGF-β1production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore,rapamycin decreased the ratio of Th17 cells/CD4 +T cells and upregulated Tregs in EAU, as detected by flow cytometry.·CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU.Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.展开更多
Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA(cDNA) microarrays were used to identify and study...Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA(cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha(a-MEM)cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol?L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase(ALP) activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction(RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology(GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta(TGF-b)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.展开更多
Estrogen plays an important role in regulating testicular Sertoli cell number. Furthermore, S-phase kinase-associated protein 2 (SKP2) plays a central role in mammalian cell cycle progression. The objective of this ...Estrogen plays an important role in regulating testicular Sertoli cell number. Furthermore, S-phase kinase-associated protein 2 (SKP2) plays a central role in mammalian cell cycle progression. The objective of this study was to determine whether 17β-estradiol can regulate the expression of SKP2, and the Sertoli cell cycle, via estrogen receptor β (ERβ), the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and extracellular signal-regulated kinase (ERK1/2) pathway. When cultured immature boar Sertoli cells were treated with 17β-estradiol, a time-dependent increase in SKP2 mRNA and protein level was observed by real-time PCR and Western blot, and 17β-estradiol activity peaked at 30 min. Treatment with ICI182780 and ERβ antagonist reduced 17β-estradiol-induced expression of SKP2 and proliferating cell nuclear antigen (PCNA), while increasing the protein concentration of p27kip1. However, the effect of ERa antagonist on these parameters was lower than that of ICI 182780 and ERβ. Forskolin had a similar effect as 17β-estradiol on the expression of SKP2, PCNA and p27kip1, Rp-cAMP, H-89 and U0126 treatment reduced 17β-estradiol-induced changes, while H-89 also inhibited ERK1/2 activation. Therefore, 17β-estradiol mainly regulates SKP2 mRNA and protein expression via ERβ-cAMP-PKA and ERK1/2 activation. SKP2 and PCNA expression were positively correlated, while increased SKP2 expression likely resulted in p27kip1 degradation.展开更多
Natural killer T cells(NKT cells) are innate-like T cells that acquire effector functions while developing in the thymus, polarize into three distinct functional subsets viz. NKT1, NKT2 and NKT17 cells that produce in...Natural killer T cells(NKT cells) are innate-like T cells that acquire effector functions while developing in the thymus, polarize into three distinct functional subsets viz. NKT1, NKT2 and NKT17 cells that produce interferon(IFN)-γ, interleukin(IL)-4 and IL-17, respectively. However, there has been no unique surface markers that define each subsets, forcing investigators to use intracellular staining of transcription factors and cytokines in combination of surface markers to distinguish among these subsets. Intracellular staining, however, causes apoptosis and prevents subsequent utilization of NKT cells in functional in vitro and in vivo assays that require viable cells. This limitation has significantly impeded understanding the specific properties of each subset and their interactions with each other. Therefore, there has been fervent efforts to find a specific markers for each NKT cell subset. We have recently identified that syndecan-1(SDC-1; CD138) as a specific surface marker of NKT17 cells. This discovery now allows visualization of NKT17 in situ and study of their peripheral tissue distribution, characteristics of their TCR and viable sorting for in vitro and in vivo analysis. In addition, it lays the ground working for investigating significance of SDC-1 expression on this particular subset in regulating their roles in host defense and glucose metabolism.展开更多
HTLV-1(human T-lymphotropic virus type 1)causes chronic infection ofhuman T lymphocytes.HTLV-1 infection is known to be related to multiple diseases,including neoplastic growth of HTLV-1-infected T cells(ATL)and neopl...HTLV-1(human T-lymphotropic virus type 1)causes chronic infection ofhuman T lymphocytes.HTLV-1 infection is known to be related to multiple diseases,including neoplastic growth of HTLV-1-infected T cells(ATL)and neoplastic inflammatory conditions,such as HTLV-1-associated myelopathy/tropical spastic paraparesis(HAM/TSP),Sjogren's syndrome with arthritis,polymyositis uveitis,and bronchoalveolitis.Regulatory T cells(Tregs)regulate inflammatory cells,such as Th17 cells.The purpose of this study was to evaluate Tregs and Th17 cells,as well as the expression of related transcription factors(ROR-γ1 and FOXP3)and cytokines(IL-10,TGF-β,IL-6,and IL-17A)in HTLV-1 infection.展开更多
Estrogen plays an important role in regulating Sertoli cell number in the testis. The objective of the study was to identify whether 17β-estradiol affected the proliferation of cultured, immature boar Sertoli cells v...Estrogen plays an important role in regulating Sertoli cell number in the testis. The objective of the study was to identify whether 17β-estradiol affected the proliferation of cultured, immature boar Sertoli cells via the estrogen receptor β (ERβ) and the cAMP-extracellular signal-regulated kinase (ERK1/2) pathway. Low levels (10-10-10-8 mol L-1) of 17β-estradiol increased cell number, but high levels (10-7-10-6 mol L-1) decreased it (P〈0.05). Sertoli cell number began to recover for an additional 24 h in the medium without 17β-estradiol (10-6 mol L-l) (P〉0.05). The effects of 17β-estradiol (10-9 mol L-1) peaked at the first 24 h (P〈0.05). 17β-estradiol activated ERK1/2 from 5 min to 24 h, but the activiy of ERK1/2 began to decrease after 4 h. Both PD98059 and U0126, two ERK inhibitors, blocked cell division (P〈0.05). 17β-estradiol (10-10-10-6 mol L-1) dose-dependently increased cAMP production (P 〈 0.05), and both 17β-estradiol (10-9 mol L-1) and forskolin, which increases cAMP levels, induced cell proliferation and activated ERK1/2 (P〈 0.05). Rp-cAMP, an antagonist of cAMP, blocked this 17β-estradiol activity (P〈 0.05). Two estrogen receptor antagonists, ICI 182780 and ERβ antagonist (ERβAnt), reduced Sertoli cell number, cAMP production and ERK1/2 activation (P〈 0.05), but ERaAnt did not (P〉 0.05). Therefore, 17β- estradiol mainly promotes pig Sertoli cell proliferation via ERβ to induce cAMP production and ERK activation to promote cell proliferation.展开更多
BACKGROUND Advanced glycation end products(AGE)are a marker of various diseases including diabetes,in which they participate to vascular damages such as retinopathy,nephropathy and coronaropathy.Besides those vascular...BACKGROUND Advanced glycation end products(AGE)are a marker of various diseases including diabetes,in which they participate to vascular damages such as retinopathy,nephropathy and coronaropathy.Besides those vascular complications,AGE are involved in altered metabolism in many tissues,including adipose tissue(AT)where they contribute to reduced glucose uptake and attenuation of insulin sensitivity.AGE are known to contribute to type 1 diabetes(T1D)through promotion of interleukin(IL)-17 secreting T helper(Th17)cells.AIM To investigate whether lean adipose-derived stem cells(ASC)could be able to induce IL-17A secretion,with the help of AGE.METHODS As we have recently demonstrated that ASC are involved in Th17 cell promotion when they are harvested from obese AT,we used the same co-culture model to measure the impact of glycated human serum albumin(G-HSA)on human lean ASC interacting with blood mononuclear cells.IL-17A and pro-inflammatory cytokine secretion were measured by ELISA.Receptor of AGE(RAGE)together with intercellular adhesion molecule 1(ICAM-1),human leukocyte Antigen(HLA)-DR,cluster of differentiation(CD)41,and CD62P surface expressions were measured by cytofluorometry.Anti-RAGE specific monoclonal antibody was added to co-cultures in order to evaluate the role of RAGE in IL-17A production.RESULTS Results showed that whereas 1%G-HSA only weakly potentiated the production of IL-17A by T cells interacting with ASC harvested from obese subjects,it markedly increased IL-17A,but also interferon gamma and tumor necrosis factor alpha production in the presence of ASC harvested from lean individuals.This was associated with increased expression of RAGE and HLA-DR molecule by cocultured cells.Moreover,RAGE blockade experiments demonstrated RAGE specific involvement in lean ASC-mediated Th-17 cell activation.Finally,platelet aggregation and ICAM-1,which are known to be induced by AGE,were not involved in these processes.CONCLUSION Thus,our results demonstrated that G-HSA potentiated lean ASC-mediated IL-17A production in AT,suggesting a new mechanism by which AGE could contribute to T1D pathophysiology.展开更多
文摘Innate-like T cells, namely natural killer T(NKT) and γδ T cells, play critical roles in linking innate and adaptive immune responses through rapid production of cytokines. Prominent among these cytokines is interleukin-17(IL-17), which is a potent proinflammatory cytokine that plays a critical role in host defense against fungi and extracellular bacteria. However, excessive IL-17-production promotes autoimmune diseases, including psoriasis, multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease, and systemic lupus erythematosus. IL-17 has also been implicated in regulating body fat, which is highly relevant given rises in obesity and type 2 diabetes. NKT cells, γδ T cells and mucosal-associated invariant T cells(MAIT) are the major sources of IL-17 involved in protection of mucosal surfaces from opportunistic infections and causing autoimmunity when become dysregulated. Given the pathogenic effects of IL-17, efforts have been directed towards understanding mechanisms that guard against IL-17 overproduction. One novel potent mechanism is mediated by the heparan sulfate proteoglycan, syndecan-1(sdc1), which is selectively expressed by IL-17-producing subsets of NKT and γδ T cells. This unexpected role for sdc1 is uncovered by analysis of NKT and γδ T cells in sdc1-deficient mice. In this mini-review, we discuss selective expression of sdc1 by these innate T cells and consequences of its absence on IL-17 homeostasis and pathological implications.
基金Supported by National Natural Science Foundation of China(No.81371005)
文摘· AIM: To determine the effects of rapamycin on experimental autoimmune uveoretinitis(EAU) and investigate of role of rapamycin on T cell subsets in the disease.·METHODS: EAU was induced in rats using peptides1169 to 1191 of the interphotoreceptor binding protein(IRBP). Rapamycin(0.2 mg/kg/d) was administrated by intraperitoneal injection for a consecutive 7d after immunization. Th1/Th2/Th17 cytokines, TGF-β1, and IL-6produced by lymphocyteswere measured by ELISA, while Th17 cells and CD4 +CD25 + regulatory T cells(Tregs)from rat spleen were detected by flow cytometry.·RESULTS: Intraperitoneal treatment immediately after immunization dramatically ameliorated the clinical course of EAU. Clinical responses were associated with reduced retinal inflammatory cell infiltration and tissue destruction. Rapamycin induced suppression of Th1/Th2/Th17 cytokines, including IFN-γ, IL-2, IL-17, IL-4, and IL-10 release from T lymphocytes of EAU rats, in vitro.Rapamycin also significantly increased TGF-β1production but had no effect on IL-6 productionof T lymphocytes from EAU rats in vitro. Furthermore,rapamycin decreased the ratio of Th17 cells/CD4 +T cells and upregulated Tregs in EAU, as detected by flow cytometry.·CONCLUSION: Rapamycin effectively interferes with T cell mediated autoimmune uveitis by inhibiting antigen-specific T cell functions and enhancing Tregs in EAU.Rapamycin is a promising new alternative as an adjunct corticosteroid-sparing agent for treating uveitis.
基金supported by grants from the Natural Science Fund (ZR2010HM035) of Shandong Provincethe Shandong Provincial Health Development Project Fund (2011WSB19002) in China
文摘Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation.In this study, complementary DNA(cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells’ response to 17-b estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha(a-MEM)cell culture supplemented with 17-b estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with1028mol?L2117-b estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase(ALP) activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction(RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5 403 differentially expressed genes,of which 1 996 genes were upregulated and 3 407 genes were downregulated, 1 553 different functional classifications were identified by gene ontology(GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta(TGF-b)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to a-MEM supplemented with 17-b estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.
基金grants from the National Natural Science Foundation of China(31072183)the Fundamental Research Funds for the Central Universities,China(XDJK2009B035)
文摘Estrogen plays an important role in regulating testicular Sertoli cell number. Furthermore, S-phase kinase-associated protein 2 (SKP2) plays a central role in mammalian cell cycle progression. The objective of this study was to determine whether 17β-estradiol can regulate the expression of SKP2, and the Sertoli cell cycle, via estrogen receptor β (ERβ), the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) and extracellular signal-regulated kinase (ERK1/2) pathway. When cultured immature boar Sertoli cells were treated with 17β-estradiol, a time-dependent increase in SKP2 mRNA and protein level was observed by real-time PCR and Western blot, and 17β-estradiol activity peaked at 30 min. Treatment with ICI182780 and ERβ antagonist reduced 17β-estradiol-induced expression of SKP2 and proliferating cell nuclear antigen (PCNA), while increasing the protein concentration of p27kip1. However, the effect of ERa antagonist on these parameters was lower than that of ICI 182780 and ERβ. Forskolin had a similar effect as 17β-estradiol on the expression of SKP2, PCNA and p27kip1, Rp-cAMP, H-89 and U0126 treatment reduced 17β-estradiol-induced changes, while H-89 also inhibited ERK1/2 activation. Therefore, 17β-estradiol mainly regulates SKP2 mRNA and protein expression via ERβ-cAMP-PKA and ERK1/2 activation. SKP2 and PCNA expression were positively correlated, while increased SKP2 expression likely resulted in p27kip1 degradation.
文摘Natural killer T cells(NKT cells) are innate-like T cells that acquire effector functions while developing in the thymus, polarize into three distinct functional subsets viz. NKT1, NKT2 and NKT17 cells that produce interferon(IFN)-γ, interleukin(IL)-4 and IL-17, respectively. However, there has been no unique surface markers that define each subsets, forcing investigators to use intracellular staining of transcription factors and cytokines in combination of surface markers to distinguish among these subsets. Intracellular staining, however, causes apoptosis and prevents subsequent utilization of NKT cells in functional in vitro and in vivo assays that require viable cells. This limitation has significantly impeded understanding the specific properties of each subset and their interactions with each other. Therefore, there has been fervent efforts to find a specific markers for each NKT cell subset. We have recently identified that syndecan-1(SDC-1; CD138) as a specific surface marker of NKT17 cells. This discovery now allows visualization of NKT17 in situ and study of their peripheral tissue distribution, characteristics of their TCR and viable sorting for in vitro and in vivo analysis. In addition, it lays the ground working for investigating significance of SDC-1 expression on this particular subset in regulating their roles in host defense and glucose metabolism.
文摘HTLV-1(human T-lymphotropic virus type 1)causes chronic infection ofhuman T lymphocytes.HTLV-1 infection is known to be related to multiple diseases,including neoplastic growth of HTLV-1-infected T cells(ATL)and neoplastic inflammatory conditions,such as HTLV-1-associated myelopathy/tropical spastic paraparesis(HAM/TSP),Sjogren's syndrome with arthritis,polymyositis uveitis,and bronchoalveolitis.Regulatory T cells(Tregs)regulate inflammatory cells,such as Th17 cells.The purpose of this study was to evaluate Tregs and Th17 cells,as well as the expression of related transcription factors(ROR-γ1 and FOXP3)and cytokines(IL-10,TGF-β,IL-6,and IL-17A)in HTLV-1 infection.
基金supported by the National Natural Science Foundation of China(30270955)the Foundamental Research Funds for the Central Universities,China(XDJK2009B035)
文摘Estrogen plays an important role in regulating Sertoli cell number in the testis. The objective of the study was to identify whether 17β-estradiol affected the proliferation of cultured, immature boar Sertoli cells via the estrogen receptor β (ERβ) and the cAMP-extracellular signal-regulated kinase (ERK1/2) pathway. Low levels (10-10-10-8 mol L-1) of 17β-estradiol increased cell number, but high levels (10-7-10-6 mol L-1) decreased it (P〈0.05). Sertoli cell number began to recover for an additional 24 h in the medium without 17β-estradiol (10-6 mol L-l) (P〉0.05). The effects of 17β-estradiol (10-9 mol L-1) peaked at the first 24 h (P〈0.05). 17β-estradiol activated ERK1/2 from 5 min to 24 h, but the activiy of ERK1/2 began to decrease after 4 h. Both PD98059 and U0126, two ERK inhibitors, blocked cell division (P〈0.05). 17β-estradiol (10-10-10-6 mol L-1) dose-dependently increased cAMP production (P 〈 0.05), and both 17β-estradiol (10-9 mol L-1) and forskolin, which increases cAMP levels, induced cell proliferation and activated ERK1/2 (P〈 0.05). Rp-cAMP, an antagonist of cAMP, blocked this 17β-estradiol activity (P〈 0.05). Two estrogen receptor antagonists, ICI 182780 and ERβ antagonist (ERβAnt), reduced Sertoli cell number, cAMP production and ERK1/2 activation (P〈 0.05), but ERaAnt did not (P〉 0.05). Therefore, 17β- estradiol mainly promotes pig Sertoli cell proliferation via ERβ to induce cAMP production and ERK activation to promote cell proliferation.
文摘BACKGROUND Advanced glycation end products(AGE)are a marker of various diseases including diabetes,in which they participate to vascular damages such as retinopathy,nephropathy and coronaropathy.Besides those vascular complications,AGE are involved in altered metabolism in many tissues,including adipose tissue(AT)where they contribute to reduced glucose uptake and attenuation of insulin sensitivity.AGE are known to contribute to type 1 diabetes(T1D)through promotion of interleukin(IL)-17 secreting T helper(Th17)cells.AIM To investigate whether lean adipose-derived stem cells(ASC)could be able to induce IL-17A secretion,with the help of AGE.METHODS As we have recently demonstrated that ASC are involved in Th17 cell promotion when they are harvested from obese AT,we used the same co-culture model to measure the impact of glycated human serum albumin(G-HSA)on human lean ASC interacting with blood mononuclear cells.IL-17A and pro-inflammatory cytokine secretion were measured by ELISA.Receptor of AGE(RAGE)together with intercellular adhesion molecule 1(ICAM-1),human leukocyte Antigen(HLA)-DR,cluster of differentiation(CD)41,and CD62P surface expressions were measured by cytofluorometry.Anti-RAGE specific monoclonal antibody was added to co-cultures in order to evaluate the role of RAGE in IL-17A production.RESULTS Results showed that whereas 1%G-HSA only weakly potentiated the production of IL-17A by T cells interacting with ASC harvested from obese subjects,it markedly increased IL-17A,but also interferon gamma and tumor necrosis factor alpha production in the presence of ASC harvested from lean individuals.This was associated with increased expression of RAGE and HLA-DR molecule by cocultured cells.Moreover,RAGE blockade experiments demonstrated RAGE specific involvement in lean ASC-mediated Th-17 cell activation.Finally,platelet aggregation and ICAM-1,which are known to be induced by AGE,were not involved in these processes.CONCLUSION Thus,our results demonstrated that G-HSA potentiated lean ASC-mediated IL-17A production in AT,suggesting a new mechanism by which AGE could contribute to T1D pathophysiology.