Chandrasekaran Raman - Academia.edu (original) (raw)

Papers by Chandrasekaran Raman

Endocrinology, 2008

Thyroid hormones require transport across cell membranes to carry out their biological functions.... more Thyroid hormones require transport across cell membranes to carry out their biological functions. The importance of transport for thyroid hormone signaling was highlighted by the discovery that inactivating mutations in the human monocarboxylate transporter-8 (MCT8) (SLC16A2) cause severe psychomotor retardation due to thyroid hormone deficiency in the central nervous system. It has been reported that Mct8 expression in the mouse brain is restricted to neurons, leading to the model that organic ion transporter polypeptide-14 (OATP14, also known as OATP1C1/SLCO1C1) is the primary thyroid hormone transporter at the blood-brain barrier, whereas MCT8 mediates thyroid hormone uptake into neurons. In contrast to these reports, we report here that in addition to neuronal expression, MCT8 mRNA and protein are expressed in cerebral microvessels in human, mouse, and rat. In addition, OATP14 mRNA and protein are strongly enriched in mouse and rat cerebral microvessels but not in human microves...

Arthritis & Rheumatism, 2001

To identify and screen the level of arthritis susceptibility in C3H murine strains known to be re... more To identify and screen the level of arthritis susceptibility in C3H murine strains known to be resistant to proteoglycan (aggrecan)-induced arthritis, and to measure and correlate various immunologic and inflammatory parameters with susceptibility to either arthritis or spondylitis in various C3H substrains. Mice of 10 C3H substrains (subcolonies) were immunized with cartilage proteoglycan (aggrecan) for induction of arthritis. Animals were assessed for clinical symptoms, and the peripheral joints and spine were studied by histologic methods. Proteoglycan-specific T cell responses (T cell proliferation and production of interleukin-2 [IL-2], interferon-y, and IL-4) and the B cell response to lipopolysaccharide (LPS) were measured in spleen cell cultures. Serum levels of heteroantibodies and autoantibodies as well as various cytokines (IL-6, IL-10, IL-12, and tumor necrosis factor alpha) and soluble CD44 were determined by enzyme-linked immunosorbent assay. Immunization with cartilage proteoglycan induced severe arthritis in the C3H/HeJCr substrain (95-100% incidence), whereas the original parent mice of the C3H/HeJ colony were resistant to proteoglycan (aggrecan)-induced arthritis. Furthermore, the progressive polyarthritis that is characteristic in susceptible C3H/HeJCr mice was accompanied by progressive inflammation around the spine. In subsequent experiments, 10 different C3H colonies with largely identical genetic backgrounds (all originating from the National Institutes of Health or Jackson Laboratory) exhibited extreme differences in susceptibility. Although none of the laboratory findings, including LPS hyporesponsiveness, immunologic parameters, and inflammatory markers, showed a correlation with susceptibility or resistance in the C3H/HeJCr and C3H/HeJ substrains, respectively, significant differences were found when all arthritic C3H mice were compared with all nonarthritic animals, regardless of their substrain origin. Because many of the C3H substrains lost arthritis susceptibility or acquired resistance, our results suggest that a preferred site for a mutation(s) in a gene(s) in a relatively upstream position of the inflammatory cascade is present. This is the first autoimmune model that exhibits extreme differences in arthritis susceptibility in the same murine strain, and is therefore a valuable tool for identification of arthritis-susceptible (or arthritis-suppressive) genes.

The Journal of Bone and Joint Surgery. British volume, 2004

Periprosthetic bone loss after total joint arthroplasty is a major clinical problem resulting in ... more Periprosthetic bone loss after total joint arthroplasty is a major clinical problem resulting in aseptic loosening of the implant. Among many cell types, osteoblasts play a crucial role in the development of peri-implant osteolysis. In this study, we tested the effects of calcitriol (1α,25-dihydroxy-vitamin-D3) and the bisphosphonate pamidronate on titanium-particle- and TNF-α-induced release of interleukin-6 and suppression of osteoblast-specific gene expressions in bone-marrow-derived stromal cells with an osteoblastic phenotype. We monitored the expression of procollagen α1[1], osteocalcin, osteonectin and alkaline phosphatase mRNAs by Northern blots and real-time reverse transcription and polymerase chain reaction analyses. The release of various cytokines was also analysed by ELISA. We found that calcitriol or pamidronate could only partially recover the altered functions of osteoblasts when added alone. Only a combination of these compounds restored all the tested functions of...

PubMed, Apr 1, 2002

Loss of heterozygosity within the short arm of chromosome 3 is a common molecular event in severa... more Loss of heterozygosity within the short arm of chromosome 3 is a common molecular event in several types of solid tumors. In breast cancer, 3p loss of heterozygosity occurs in invasive tumor cells as well as in morphologically normal terminal ductal lobular units adjacent to carcinoma in some cases [G. Deng et al., Science (Wash. DC), 274: 2057-2059, 1996.]. The most frequent region of allelic loss at 3p24.3 in morphologically normal terminal ductal lobular units encompasses the thyroid hormone receptor beta1 (TRbeta1) gene. Here we have observed a variable degree of TRbeta1 promoter hypermethylation in all 11 cases of primary breast cancer examined. Moreover, hypermethylation occurred at the same CpG sites in nonmalignant tissue peripheral to carcinoma in 4 of 11 cases. The lack of TRbeta1 nuclear staining, a likely result of biallelic gene inactivation, was observed in 25% (22 of 85) of primary tumors. This is a first demonstration of promoter hypermethylation and a concurrent reduction of TRbeta1 transcripts in breast cancer cell lines, although specific CpG sites targeted for gene silencing remain to be determined. Gene expression was restored by treatment with 5-aza-deoxycytidine in such cases. The observation of early, frequent, and multiple mechanisms of TRbeta1 inactivation suggests a potential role for this gene in the suppression of breast tumorigenesis.

Background: Particle-challenged cells release cytokines, chemokines, and eicosanoids, which contr... more Background: Particle-challenged cells release cytokines, chemokines, and eicosanoids, which contribute to periprosthetic osteolysis. The particle-induced activation of macrophages and monocytes has been extensively studied, but only limited information is available on the response of osteoblasts to particulate wear debris. This study examines the effects of particulate wear debris, proinflammatory cytokines, and growth factors on osteoblast functions. Methods: MG-63 osteoblasts were treated with metal particles (titanium, titanium alloy, and chromium orthophosphate) or polymeric particles (polyethylene and polystyrene) of phagocytosable sizes or were treated with exogenous cytokines and growth factors. The kinetics of particle phagocytosis and the number of engulfed particles were assessed with use of fluoresceinated particles. Cell proliferation was determined according to [3H]-thymidine incorporation, and cell viability was determined by either fluorescein diacetate uptake or tryp...

BALB/c mice immunized with human cartilage proteoglycan (PG) develop arthritis accompanied by the... more BALB/c mice immunized with human cartilage proteoglycan (PG) develop arthritis accompanied by the production of autoantibodies to mouse cartilage PG. To determine whether the autoantibody isotype contributes to the onset and severity of arthritis, PG-specific serum IgG1 (Th2, IL-4-cytokinesupporting) and IgG2a (Th1, IFN-γ-controlling) concentrations were monitored during immunization with PG in IL-4-deficient and IFN-γ-deficient mice. Paradoxically, despite elevated IFN-γ, the PGspecific IgG1 isotype was significantly higher than the PG-specific IgG2a response, and the PGspecific IgG1 isotype was independent of IL-4. In contrast, the serum concentration of PG-specific IgG2a isotype was six times higher in IL-4-deficient mice than in wild-type controls. Moreover, the high concentration of PG-specific IgG2a isotype in IL-4-deficient mice corresponded to an increased severity of arthritis. The concentration of PG-specific IgG2a isotype was lower in IFN-γ-deficient mice than in wild-type mice, and the incidence and severity of arthritis also were significantly lower. Concentrations of PG-specific IgG2a isotype autoantibody correlated with the onset and severity of arthritis, suggesting a pathological role of this isotype, probably locally in the joint.

Endocrinology, 2008

Thyroid hormones require transport across cell membranes in order to carry out their biological f... more Thyroid hormones require transport across cell membranes in order to carry out their biological functions. The importance of transport for thyroid hormone signaling was highlighted by the discovery that inactivating mutations in the human MCT8 transporter (SLC16A2) cause severe psychomotor retardation due to thyroid hormone deficiency in the central nervous system. It has been reported that Mct8 expression in the mouse brain is restricted to neurons, leading to the model that OATP14 (OATP1C1, SLCO1C1) is the primary thyroid hormone transporter at the blood brain barrier, while MCT8 mediates thyroid hormone uptake into neurons. In contrast to these reports, we report here that in addition to neuronal expression, MCT8 mRNA and protein are expressed in cerebral microvessels in human, mouse, and rat. In addition, OATP14 mRNA and protein was strongly enriched in mouse and rat cerebral microvessels, but not in human microvessels. In rat, Mct8 and Oatp14 proteins localize to both the luminal and abluminal microvessel membranes. In human and rodent choroid plexus epithelial cells, MCT8 is concentrated on the epithelial cell apical surface and OATP14 localizes primarily to the basallateral surface. Mct8 and Oatp14 expression was also observed in mouse and rat tanycytes, which are thought to form a barrier between hypothalamic blood vessels and brain. These results raise the possibility that reduced thyroid hormone transport across the blood-brain barrier contributes to the neurological deficits observed in affected patients with MCT8 mutations. The high microvessel expression of OATP14 in rodent compared to human brain may contribute to the relatively mild phenotype observed in Mct8-null mice, in contrast to humans lacking functional MCT8.

Arthritis & Rheumatism, 2001

To identify and screen the level of arthritis susceptibility in C3H murine strains known to be re... more To identify and screen the level of arthritis susceptibility in C3H murine strains known to be resistant to proteoglycan (aggrecan)-induced arthritis, and to measure and correlate various immunologic and inflammatory parameters with susceptibility to either arthritis or spondylitis in various C3H substrains. Mice of 10 C3H substrains (subcolonies) were immunized with cartilage proteoglycan (aggrecan) for induction of arthritis. Animals were assessed for clinical symptoms, and the peripheral joints and spine were studied by histologic methods. Proteoglycan-specific T cell responses (T cell proliferation and production of interleukin-2 [IL-2], interferon-y, and IL-4) and the B cell response to lipopolysaccharide (LPS) were measured in spleen cell cultures. Serum levels of heteroantibodies and autoantibodies as well as various cytokines (IL-6, IL-10, IL-12, and tumor necrosis factor alpha) and soluble CD44 were determined by enzyme-linked immunosorbent assay. Immunization with cartilage proteoglycan induced severe arthritis in the C3H/HeJCr substrain (95-100% incidence), whereas the original parent mice of the C3H/HeJ colony were resistant to proteoglycan (aggrecan)-induced arthritis. Furthermore, the progressive polyarthritis that is characteristic in susceptible C3H/HeJCr mice was accompanied by progressive inflammation around the spine. In subsequent experiments, 10 different C3H colonies with largely identical genetic backgrounds (all originating from the National Institutes of Health or Jackson Laboratory) exhibited extreme differences in susceptibility. Although none of the laboratory findings, including LPS hyporesponsiveness, immunologic parameters, and inflammatory markers, showed a correlation with susceptibility or resistance in the C3H/HeJCr and C3H/HeJ substrains, respectively, significant differences were found when all arthritic C3H mice were compared with all nonarthritic animals, regardless of their substrain origin. Because many of the C3H substrains lost arthritis susceptibility or acquired resistance, our results suggest that a preferred site for a mutation(s) in a gene(s) in a relatively upstream position of the inflammatory cascade is present. This is the first autoimmune model that exhibits extreme differences in arthritis susceptibility in the same murine strain, and is therefore a valuable tool for identification of arthritis-susceptible (or arthritis-suppressive) genes.

The Journal of Bone and Joint Surgery, 2004

Arthritis and Rheumatism, Jun 1, 2003

Objective. To explore the effect of sex on clinical and immunologic traits in major histocompatib... more Objective. To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex-matched (H-2d) F 2 hybrid mice with proteoglycan (PG)-induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome. Methods. (BALB/c ؋ DBA/2)F 2 hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using >200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor ␣, interleukin-1 [IL-1], IL-6, interferon-␥, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F 2 hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed. Results. Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus-locus interaction. Conclusion. Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex. Rheumatoid arthritis (RA) is a complex inflammatory autoimmune disease with a female prevalence that affects ϳ1% of the population (1-4). In spite of extensive epidemiologic, genetic, and immunologic studies, the etiology of RA remains unknown. Because of the difficulty of examining early events in disease progression, most studies concentrate on inflammation in peripheral joints when and where the clinical symptoms become evident. Linkage, or quantitative trait loci (QTL), analysis is a unique method of identifying susceptibility genes, at least theoretically, irrespective of the time point at which the gene was, or is still, active. Linkage analysis of families with RA probands is in progress, and several chromosomal regions (QTLs) have already been identified (5-14). Among the diseaseassociated loci, the major histocompatibility complex (MHC) on human chromosome 6 has the strongest effect on RA susceptibility, as it does in many other autoimmune diseases (5,6,11,12,14,15).

Journal of Bone and Mineral Research, 2000

Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a var... more Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

Neuroscience, 2008

Nutrient transporters and ABC efflux pumps at the blood–brain barrier are major determinants of d... more Nutrient transporters and ABC efflux pumps at the blood–brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel

Amyotrophic Lateral Sclerosis, 2004

Effective treatment for amyotrophic lateral sclerosis (ALS) remains elusive. Two of the primary h... more Effective treatment for amyotrophic lateral sclerosis (ALS) remains elusive. Two of the primary hypothesesunderlying motor neuron vulnerability are susceptibility to excitotoxi- city and oxidative damage. There is rapidly emerging evidence that the cannabinoid receptor system has the potential to reduce both excitotoxic and oxidative cell damage. Here we report that treatment with D 9 - tetrahydrocannabinol (D 9 -THC) was

The Journal of Immunology, Jul 1, 2001

CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion rece... more CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion receptor for the extracellular matrix glycosaminoglycan hyaluronan. CD44 participates in a variety of physiological and pathological processes through its role in cell adhesion. Under appropriate conditions, the ectodomain of CD44 is proteolytically removed from the cell surface. In this study we show that excessive CD44 shedding can be induced in mouse fibroblasts and monocytes upon exposure of these cells to a CD44specific Ab immobilized on plastic, whereas treatment with phorbol ester induces significantly enhanced CD44 release from the monocytes only. CD44 shedding proceeds normally in fibroblasts and monocytes deficient in TNF-␣ converting enzyme (TACE), a sheddase involved in the processing of several substrates. Conversely, activation of the CD44 protease has no effect on the release of TNF-␣ from TACE-expressing cells, although the same metalloprotease inhibitor effectively blocks both TACE and the CD44 sheddase. Concomitant with anti-CD44 Ab-or phorbol ester-induced CD44 shedding, dramatic changes are observed in cell morphology and the structure of the actin cytoskeleton. Disruption of actin assembly with cytochalasin reduces CD44 shedding, but not the release of TNF-␣. Moreover, pharmacological activation of Rho family GTPases Rac1 and Cdc42, which regulate actin filament assembly into distinct cytoskeletal structures, has a profound effect on CD44 release. We conclude that the CD44 sheddase and TACE are distinct enzymes, and that Ab-and phorbol ester-enhanced cleavage of CD44 is controlled in a cell type-dependent fashion by Rho GTPases through the cytoskeleton.

Endocrinology, 2008

Thyroid hormones require transport across cell membranes to carry out their biological functions.... more Thyroid hormones require transport across cell membranes to carry out their biological functions. The importance of transport for thyroid hormone signaling was highlighted by the discovery that inactivating mutations in the human monocarboxylate transporter-8 (MCT8) (SLC16A2) cause severe psychomotor retardation due to thyroid hormone deficiency in the central nervous system. It has been reported that Mct8 expression in the mouse brain is restricted to neurons, leading to the model that organic ion transporter polypeptide-14 (OATP14, also known as OATP1C1/SLCO1C1) is the primary thyroid hormone transporter at the blood-brain barrier, whereas MCT8 mediates thyroid hormone uptake into neurons. In contrast to these reports, we report here that in addition to neuronal expression, MCT8 mRNA and protein are expressed in cerebral microvessels in human, mouse, and rat. In addition, OATP14 mRNA and protein are strongly enriched in mouse and rat cerebral microvessels but not in human microves...

Arthritis & Rheumatism, 2001

To identify and screen the level of arthritis susceptibility in C3H murine strains known to be re... more To identify and screen the level of arthritis susceptibility in C3H murine strains known to be resistant to proteoglycan (aggrecan)-induced arthritis, and to measure and correlate various immunologic and inflammatory parameters with susceptibility to either arthritis or spondylitis in various C3H substrains. Mice of 10 C3H substrains (subcolonies) were immunized with cartilage proteoglycan (aggrecan) for induction of arthritis. Animals were assessed for clinical symptoms, and the peripheral joints and spine were studied by histologic methods. Proteoglycan-specific T cell responses (T cell proliferation and production of interleukin-2 [IL-2], interferon-y, and IL-4) and the B cell response to lipopolysaccharide (LPS) were measured in spleen cell cultures. Serum levels of heteroantibodies and autoantibodies as well as various cytokines (IL-6, IL-10, IL-12, and tumor necrosis factor alpha) and soluble CD44 were determined by enzyme-linked immunosorbent assay. Immunization with cartilage proteoglycan induced severe arthritis in the C3H/HeJCr substrain (95-100% incidence), whereas the original parent mice of the C3H/HeJ colony were resistant to proteoglycan (aggrecan)-induced arthritis. Furthermore, the progressive polyarthritis that is characteristic in susceptible C3H/HeJCr mice was accompanied by progressive inflammation around the spine. In subsequent experiments, 10 different C3H colonies with largely identical genetic backgrounds (all originating from the National Institutes of Health or Jackson Laboratory) exhibited extreme differences in susceptibility. Although none of the laboratory findings, including LPS hyporesponsiveness, immunologic parameters, and inflammatory markers, showed a correlation with susceptibility or resistance in the C3H/HeJCr and C3H/HeJ substrains, respectively, significant differences were found when all arthritic C3H mice were compared with all nonarthritic animals, regardless of their substrain origin. Because many of the C3H substrains lost arthritis susceptibility or acquired resistance, our results suggest that a preferred site for a mutation(s) in a gene(s) in a relatively upstream position of the inflammatory cascade is present. This is the first autoimmune model that exhibits extreme differences in arthritis susceptibility in the same murine strain, and is therefore a valuable tool for identification of arthritis-susceptible (or arthritis-suppressive) genes.

The Journal of Bone and Joint Surgery. British volume, 2004

Periprosthetic bone loss after total joint arthroplasty is a major clinical problem resulting in ... more Periprosthetic bone loss after total joint arthroplasty is a major clinical problem resulting in aseptic loosening of the implant. Among many cell types, osteoblasts play a crucial role in the development of peri-implant osteolysis. In this study, we tested the effects of calcitriol (1α,25-dihydroxy-vitamin-D3) and the bisphosphonate pamidronate on titanium-particle- and TNF-α-induced release of interleukin-6 and suppression of osteoblast-specific gene expressions in bone-marrow-derived stromal cells with an osteoblastic phenotype. We monitored the expression of procollagen α1[1], osteocalcin, osteonectin and alkaline phosphatase mRNAs by Northern blots and real-time reverse transcription and polymerase chain reaction analyses. The release of various cytokines was also analysed by ELISA. We found that calcitriol or pamidronate could only partially recover the altered functions of osteoblasts when added alone. Only a combination of these compounds restored all the tested functions of...

PubMed, Apr 1, 2002

Loss of heterozygosity within the short arm of chromosome 3 is a common molecular event in severa... more Loss of heterozygosity within the short arm of chromosome 3 is a common molecular event in several types of solid tumors. In breast cancer, 3p loss of heterozygosity occurs in invasive tumor cells as well as in morphologically normal terminal ductal lobular units adjacent to carcinoma in some cases [G. Deng et al., Science (Wash. DC), 274: 2057-2059, 1996.]. The most frequent region of allelic loss at 3p24.3 in morphologically normal terminal ductal lobular units encompasses the thyroid hormone receptor beta1 (TRbeta1) gene. Here we have observed a variable degree of TRbeta1 promoter hypermethylation in all 11 cases of primary breast cancer examined. Moreover, hypermethylation occurred at the same CpG sites in nonmalignant tissue peripheral to carcinoma in 4 of 11 cases. The lack of TRbeta1 nuclear staining, a likely result of biallelic gene inactivation, was observed in 25% (22 of 85) of primary tumors. This is a first demonstration of promoter hypermethylation and a concurrent reduction of TRbeta1 transcripts in breast cancer cell lines, although specific CpG sites targeted for gene silencing remain to be determined. Gene expression was restored by treatment with 5-aza-deoxycytidine in such cases. The observation of early, frequent, and multiple mechanisms of TRbeta1 inactivation suggests a potential role for this gene in the suppression of breast tumorigenesis.

Background: Particle-challenged cells release cytokines, chemokines, and eicosanoids, which contr... more Background: Particle-challenged cells release cytokines, chemokines, and eicosanoids, which contribute to periprosthetic osteolysis. The particle-induced activation of macrophages and monocytes has been extensively studied, but only limited information is available on the response of osteoblasts to particulate wear debris. This study examines the effects of particulate wear debris, proinflammatory cytokines, and growth factors on osteoblast functions. Methods: MG-63 osteoblasts were treated with metal particles (titanium, titanium alloy, and chromium orthophosphate) or polymeric particles (polyethylene and polystyrene) of phagocytosable sizes or were treated with exogenous cytokines and growth factors. The kinetics of particle phagocytosis and the number of engulfed particles were assessed with use of fluoresceinated particles. Cell proliferation was determined according to [3H]-thymidine incorporation, and cell viability was determined by either fluorescein diacetate uptake or tryp...

BALB/c mice immunized with human cartilage proteoglycan (PG) develop arthritis accompanied by the... more BALB/c mice immunized with human cartilage proteoglycan (PG) develop arthritis accompanied by the production of autoantibodies to mouse cartilage PG. To determine whether the autoantibody isotype contributes to the onset and severity of arthritis, PG-specific serum IgG1 (Th2, IL-4-cytokinesupporting) and IgG2a (Th1, IFN-γ-controlling) concentrations were monitored during immunization with PG in IL-4-deficient and IFN-γ-deficient mice. Paradoxically, despite elevated IFN-γ, the PGspecific IgG1 isotype was significantly higher than the PG-specific IgG2a response, and the PGspecific IgG1 isotype was independent of IL-4. In contrast, the serum concentration of PG-specific IgG2a isotype was six times higher in IL-4-deficient mice than in wild-type controls. Moreover, the high concentration of PG-specific IgG2a isotype in IL-4-deficient mice corresponded to an increased severity of arthritis. The concentration of PG-specific IgG2a isotype was lower in IFN-γ-deficient mice than in wild-type mice, and the incidence and severity of arthritis also were significantly lower. Concentrations of PG-specific IgG2a isotype autoantibody correlated with the onset and severity of arthritis, suggesting a pathological role of this isotype, probably locally in the joint.

Endocrinology, 2008

Thyroid hormones require transport across cell membranes in order to carry out their biological f... more Thyroid hormones require transport across cell membranes in order to carry out their biological functions. The importance of transport for thyroid hormone signaling was highlighted by the discovery that inactivating mutations in the human MCT8 transporter (SLC16A2) cause severe psychomotor retardation due to thyroid hormone deficiency in the central nervous system. It has been reported that Mct8 expression in the mouse brain is restricted to neurons, leading to the model that OATP14 (OATP1C1, SLCO1C1) is the primary thyroid hormone transporter at the blood brain barrier, while MCT8 mediates thyroid hormone uptake into neurons. In contrast to these reports, we report here that in addition to neuronal expression, MCT8 mRNA and protein are expressed in cerebral microvessels in human, mouse, and rat. In addition, OATP14 mRNA and protein was strongly enriched in mouse and rat cerebral microvessels, but not in human microvessels. In rat, Mct8 and Oatp14 proteins localize to both the luminal and abluminal microvessel membranes. In human and rodent choroid plexus epithelial cells, MCT8 is concentrated on the epithelial cell apical surface and OATP14 localizes primarily to the basallateral surface. Mct8 and Oatp14 expression was also observed in mouse and rat tanycytes, which are thought to form a barrier between hypothalamic blood vessels and brain. These results raise the possibility that reduced thyroid hormone transport across the blood-brain barrier contributes to the neurological deficits observed in affected patients with MCT8 mutations. The high microvessel expression of OATP14 in rodent compared to human brain may contribute to the relatively mild phenotype observed in Mct8-null mice, in contrast to humans lacking functional MCT8.

Arthritis & Rheumatism, 2001

To identify and screen the level of arthritis susceptibility in C3H murine strains known to be re... more To identify and screen the level of arthritis susceptibility in C3H murine strains known to be resistant to proteoglycan (aggrecan)-induced arthritis, and to measure and correlate various immunologic and inflammatory parameters with susceptibility to either arthritis or spondylitis in various C3H substrains. Mice of 10 C3H substrains (subcolonies) were immunized with cartilage proteoglycan (aggrecan) for induction of arthritis. Animals were assessed for clinical symptoms, and the peripheral joints and spine were studied by histologic methods. Proteoglycan-specific T cell responses (T cell proliferation and production of interleukin-2 [IL-2], interferon-y, and IL-4) and the B cell response to lipopolysaccharide (LPS) were measured in spleen cell cultures. Serum levels of heteroantibodies and autoantibodies as well as various cytokines (IL-6, IL-10, IL-12, and tumor necrosis factor alpha) and soluble CD44 were determined by enzyme-linked immunosorbent assay. Immunization with cartilage proteoglycan induced severe arthritis in the C3H/HeJCr substrain (95-100% incidence), whereas the original parent mice of the C3H/HeJ colony were resistant to proteoglycan (aggrecan)-induced arthritis. Furthermore, the progressive polyarthritis that is characteristic in susceptible C3H/HeJCr mice was accompanied by progressive inflammation around the spine. In subsequent experiments, 10 different C3H colonies with largely identical genetic backgrounds (all originating from the National Institutes of Health or Jackson Laboratory) exhibited extreme differences in susceptibility. Although none of the laboratory findings, including LPS hyporesponsiveness, immunologic parameters, and inflammatory markers, showed a correlation with susceptibility or resistance in the C3H/HeJCr and C3H/HeJ substrains, respectively, significant differences were found when all arthritic C3H mice were compared with all nonarthritic animals, regardless of their substrain origin. Because many of the C3H substrains lost arthritis susceptibility or acquired resistance, our results suggest that a preferred site for a mutation(s) in a gene(s) in a relatively upstream position of the inflammatory cascade is present. This is the first autoimmune model that exhibits extreme differences in arthritis susceptibility in the same murine strain, and is therefore a valuable tool for identification of arthritis-susceptible (or arthritis-suppressive) genes.

The Journal of Bone and Joint Surgery, 2004

Arthritis and Rheumatism, Jun 1, 2003

Objective. To explore the effect of sex on clinical and immunologic traits in major histocompatib... more Objective. To explore the effect of sex on clinical and immunologic traits in major histocompatibility complex-matched (H-2d) F 2 hybrid mice with proteoglycan (PG)-induced arthritis and to identify how the quantitative trait locus (QTL) on the X chromosome influences the onset QTL of another chromosome. Methods. (BALB/c ؋ DBA/2)F 2 hybrid mice were immunized with cartilage PG, and a genome-wide linkage analysis was performed using >200 simple sequence-length polymorphic markers. The major clinical traits (susceptibility, onset, and severity) were assessed, and PG-specific T and B cell responses, and the production of proinflammatory and antiinflammatory cytokines (tumor necrosis factor ␣, interleukin-1 [IL-1], IL-6, interferon-␥, IL-4, IL-10, and IL-12) were measured in 133 arthritic and 426 nonarthritic female and male F 2 hybrid mice. The major clinical and immunologic traits were linked to genetic loci, and potential linkages among these QTLs and the effect of sex were analyzed. Results. Thirteen QTLs reported in previous studies were confirmed. Binary traits (susceptibility to arthritis) and disease onset were female specific and were identified on chromosomes 3, 7, 10, 11, 13, and X. QTLs for disease severity were mostly male specific and were located on chromosomes 1, 4, 5, 8, 14, 15, and 19. In addition, we identified 4 new QTLs for the onset of arthritis on chromosomes 3, 4, and 11, and 1 new QTL for severity on chromosome 14; all showed a strong gender association. A locus on the X chromosome interacted with a QTL on chromosome 10, and these 2 loci together seemed to control disease incidence and onset. Most of the clinical traits (QTLs) shared common regions with the immunologic traits and frequently showed a locus-locus interaction. Conclusion. Numerous immunologic QTLs overlap with clinical QTLs, thus providing information about possible mechanisms underlying QTL function. Disease susceptibility and onset showed predominant linkage with the female sex, under the control of a QTL on the X chromosome, while the severity QTLs were more strongly linked to the male sex. Rheumatoid arthritis (RA) is a complex inflammatory autoimmune disease with a female prevalence that affects ϳ1% of the population (1-4). In spite of extensive epidemiologic, genetic, and immunologic studies, the etiology of RA remains unknown. Because of the difficulty of examining early events in disease progression, most studies concentrate on inflammation in peripheral joints when and where the clinical symptoms become evident. Linkage, or quantitative trait loci (QTL), analysis is a unique method of identifying susceptibility genes, at least theoretically, irrespective of the time point at which the gene was, or is still, active. Linkage analysis of families with RA probands is in progress, and several chromosomal regions (QTLs) have already been identified (5-14). Among the diseaseassociated loci, the major histocompatibility complex (MHC) on human chromosome 6 has the strongest effect on RA susceptibility, as it does in many other autoimmune diseases (5,6,11,12,14,15).

Journal of Bone and Mineral Research, 2000

Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a var... more Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

Neuroscience, 2008

Nutrient transporters and ABC efflux pumps at the blood–brain barrier are major determinants of d... more Nutrient transporters and ABC efflux pumps at the blood–brain barrier are major determinants of drug penetration into the brain. Immunohistochemical analysis of transporter subcellular localization is challenging due to the close apposition of the luminal and abluminal microvessel plasma membranes. We employed in vivo perfusion of biotinylation reagent through rat brain microvessels to domain-specifically label proteins exposed on the microvessel

Amyotrophic Lateral Sclerosis, 2004

Effective treatment for amyotrophic lateral sclerosis (ALS) remains elusive. Two of the primary h... more Effective treatment for amyotrophic lateral sclerosis (ALS) remains elusive. Two of the primary hypothesesunderlying motor neuron vulnerability are susceptibility to excitotoxi- city and oxidative damage. There is rapidly emerging evidence that the cannabinoid receptor system has the potential to reduce both excitotoxic and oxidative cell damage. Here we report that treatment with D 9 - tetrahydrocannabinol (D 9 -THC) was

The Journal of Immunology, Jul 1, 2001

CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion rece... more CD44 is a widely expressed integral membrane glycoprotein that serves as a specific adhesion receptor for the extracellular matrix glycosaminoglycan hyaluronan. CD44 participates in a variety of physiological and pathological processes through its role in cell adhesion. Under appropriate conditions, the ectodomain of CD44 is proteolytically removed from the cell surface. In this study we show that excessive CD44 shedding can be induced in mouse fibroblasts and monocytes upon exposure of these cells to a CD44specific Ab immobilized on plastic, whereas treatment with phorbol ester induces significantly enhanced CD44 release from the monocytes only. CD44 shedding proceeds normally in fibroblasts and monocytes deficient in TNF-␣ converting enzyme (TACE), a sheddase involved in the processing of several substrates. Conversely, activation of the CD44 protease has no effect on the release of TNF-␣ from TACE-expressing cells, although the same metalloprotease inhibitor effectively blocks both TACE and the CD44 sheddase. Concomitant with anti-CD44 Ab-or phorbol ester-induced CD44 shedding, dramatic changes are observed in cell morphology and the structure of the actin cytoskeleton. Disruption of actin assembly with cytochalasin reduces CD44 shedding, but not the release of TNF-␣. Moreover, pharmacological activation of Rho family GTPases Rac1 and Cdc42, which regulate actin filament assembly into distinct cytoskeletal structures, has a profound effect on CD44 release. We conclude that the CD44 sheddase and TACE are distinct enzymes, and that Ab-and phorbol ester-enhanced cleavage of CD44 is controlled in a cell type-dependent fashion by Rho GTPases through the cytoskeleton.