Osteoarthritis Year in Review 2016: biomarkers (biochemical markers).

PURPOSE: The aim of this "Year in Review" article is to summarize and discuss the implications of biochemical marker related articles published between the Osteoarthritis Research Society International (OARSI) 2015 Congress in Seattle and the OARSI 2016 Congress in Amsterdam. METHODS: The PubMed/MEDLINE bibliographic database was searched using the combined keywords: 'biomarker' and 'osteoarthritis'. The PubMed/MEDLINE literature search was conducted using the Advanced Search Builder function (http://www.ncbi.nlm.nih.gov/pubmed/advanced). RESULTS: Over two hundred new biomarker-related papers were published during the literature search period. Some papers identified new biomarkers whereas others explored the biological properties and clinical utility of existing markers. There were specific references to several adipocytokines including leptin and adiponectin. ADAM Metallopeptidase with Thrombospondin Type 1 motif 4 (ADAMTS-4) and aggrecan ARGS neo-epitope fragment (ARGS) in synovial fluid (SF) and plasma chemokine (CeC motif) ligand 3 (CCL3) were reported as potential new knee biomarkers. New and refined proteomic technologies and novel assays including a fluoro-microbead guiding chip (FMGC) for measuring C-telopeptide of type II collagen (CTX-II) in serum and urine and a novel magnetic nanoparticle-based technology (termed magnetic capture) for collecting and concentrating CTX-II, were described this past year. CONCLUSION: There has been steady progress in osteoarthritis (OA) biomarker research in 2016. Several novel biomarkers were identified and new technologies have been developed for measuring existing biomarkers. However, there has been no "quantum leap" this past year and identification of novel early OA biomarkers remains challenging. During the past year, OARSI published a set of recommendations for the use of soluble biomarkers in clinical trials, which is a major step forward in the clinical use of OA biomarkers and bodes well for future OA biomarker development.

Building a multidisciplinary team for burn treatment - Lessons learned from the Montreal tendon transfer experience.

Multidisciplinary teams (MDTs) represent a recognized component of care in the treatment of complex conditions such as burns. However, most institutions do not provide adequate support for the formation of these teams. Furthermore, the majority of specialists lack the managerial skills required to create a team and have difficulties finding the proper tools. Our objective is to provide an insight for health care professionals, who wish to form a MDT for burn treatment, on the challenges that are likely to be faced, and to identify key elements that may facilitate the establishment of such a project. The setting for this was a plastic surgery department and rehabilitation center at a national reference center. A qualitative analysis was performed on all correspondences related to our tetraplegia project, from 2006 to 2008. To guide our thematic analysis, we used a form of systems theory known as the complexity theory. The qualitative analysis was performed using the NVivo software (Version 8.0 QSR International Melbourne, Australia). Lastly, the data was organized in chronologic order. Three main themes emerged from the results: knowledge acquisition, project organizational setup and project steps design. These themes represented respectively 24%, 50% and 26% of all correspondences. Project steps design and knowledge acquisition correspondences increased significantly after the introduction of the mentor team to our network. We conclude that an early association with a mentor team is beneficial for the establishment of a MDT.

Les équipes multidisciplinaires (EMD) représentent une composante des soins reconnue dans le traitement de conditions complexes telles que les brûlures. Cependant, la plupart des institutions ne fournissent pas de soutien adéquat pour la formation de ces équipes. En outre, la majorité des spécialistes ne possède pas les compétences de gestion nécessaires pour créer une équipe et éprouvent souvent des difficultés à trouver les outils appropriés. Notre objectif est de fournir aux professionnels de soins de santé, qui souhaitent former une équipe multidisciplinaire pour le traitement des brûlures, un aperçu sur les défis susceptibles d'être confronter et d'identifier les éléments clés qui faciliteront la mise en place d'un tel projet. Cette étude a eu lieu dans un département de chirurgie plastique et un centre de réadaptation affilié à un centre de référence national. Une analyse qualitative a été effectuée sur toutes les correspondances relatives à notre projet de tétraplégie, de 2006 à 2008. Pour guider notre analyse thématique, nous avons utilisé une forme de la théorie des systèmes connu comme la théorie de la complexité. L'analyse qualitative a été réalisée en utilisant le logiciel NVivo (version 8.0 QSR International, Melbourne, Australie). Enfin, les données ont été organisées en ordre chronologique. Trois thèmes principaux ont émergé à partir des résultats: l'acquisition de connaissances, la configuration et l'organisation du projet ainsi que la conception des étapes du projet. Ces thèmes représentaient respectivement 24 %, 50 % et 26 % de toutes les correspondances. Les correspondances en lien avec la conception du projet et l'acquisition des connaissances ont considérablement augmenté suite à l'introduction de l'équipe de mentors à notre réseau. Nous concluons qu'une association précoce avec une équipe de mentors est bénéfique à la mise en place d'une équipe multidisciplinaire.

The effect of targeted rheumatoid arthritis therapies on anti-citrullinated protein autoantibody levels and B cell responses.

Rheumatoid arthritis (RA) is a complex inflammatory disorder associated with synovitis and joint destruction that affects an estimated 1·3 million Americans and causes significant morbidity, a reduced life-span and lost work productivity. The use of biological therapies for the treatment of RA is costly, and the selection of therapies is still largely empirical and not guided by the underlying biological features of the disease in individual patients. The synovitis associated with RA is characterized by an influx of B and T cells, macrophages and neutrophils and the expansion of fibroblast-like synoviocytes, which form pannus and lead to cartilage and bone destruction. RA is associated with synovial production of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) and with the production of inflammatory cytokines, including interleukin (IL)-1, IL-6, IL-17 and tumour necrosis factor (TNF)-α, which are targets for RA therapeutics. Recent ideas about the pathogenesis of RA emphasize a genetic predisposition to develop RA, a preclinical phase of disease that is associated with the production of ACPA and the development of symptomatic disease following inflammatory initiating events that are associated with expression of citrullinated epitopes in the joints of patients. However, we still have a limited understanding of the cytokine and intracellular pathways that regulate ACPA levels. In humans, therapy with biological agents affords a unique opportunity to better understand the cytokine and signalling pathways regulating ACPA levels and the impact of ACPA level changes on disease activity. In this study we summarize the effect of RA therapies on ACPA levels and B cell responses.

Immunophenotypic and gene expression analysis of monoclonal B-cell lymphocytosis shows biologic characteristics associated with good prognosis CLL.

Monoclonal B-cell lymphocytosis (MBL) is a hematologic condition wherein small B-cell clones can be detected in the blood of asymptomatic individuals. Most MBL have an immunophenotype similar to chronic lymphocytic leukemia (CLL), and 'CLL-like' MBL is a precursor to CLL. We used flow cytometry to identify MBL from unaffected members of CLL kindreds. We identified 101 MBL cases from 622 study subjects; of these, 82 individuals with MBL were further characterized. In all, 91 unique MBL clones were detected: 73 CLL-like MBL (CD5(+)CD20(dim)sIg(dim)), 11 atypical MBL (CD5(+)CD20(+)sIg(+)) and 7 CD5(neg) MBL (CD5(neg)CD20(+)sIg(neg)). Extended immunophenotypic characterization of these MBL subtypes was performed, and significant differences in cell surface expression of CD23, CD49d, CD79b and FMC-7 were observed among the groups. Markers of risk in CLL such as CD38, ZAP70 and CD49d were infrequently expressed in CLL-like MBL, but were expressed in the majority of atypical MBL. Interphase cytogenetics was performed in 35 MBL cases, and del 13q14 was most common (22/30 CLL-like MBL cases). Gene expression analysis using oligonucleotide arrays was performed on seven CLL-like MBL, and showed activation of B-cell receptor associated pathways. Our findings underscore the diversity of MBL subtypes and further clarify the relationship between MBL and other lymphoproliferative disorders.

Single-cell analysis reveals oligoclonality among 'low-count' monoclonal B-cell lymphocytosis.

Monoclonal B-cell lymphocytosis (MBL) is a preclinical hematologic syndrome characterized by small accumulations of CD5(+) B lymphocytes. Most MBL share phenotypic characteristics with chronic lymphocytic leukemia (CLL). Although some MBL progress to CLL, most MBL have apparently limited potential for progression to CLL, particularly those MBL with normal absolute B-cell counts ('low-count' MBL). Most CLL are monoclonal and it is not known whether MBL are monoclonal or oligoclonal; this is important because it is unclear whether MBL represent indolent CLL or represent a distinct premalignant precursor before the development of CLL. We used flow cytometry analysis and sorting to determine immunophenotypic characteristics, clonality and molecular features of MBL from familial CLL kindreds. Single-cell analysis indicated four of six low-count MBL consisted of two or more unrelated clones; the other two MBL were monoclonal. 87% of low-count MBL clones had mutated immunoglobulin genes, and no immunoglobulin heavy-chain rearrangements of V(H) family 1 were observed. Some MBL were diversified, clonally related populations with evidence of antigen drive. We conclude that although low-count MBL share many phenotypic characteristics with CLL, many MBL are oligoclonal. This supports a model for step-wise development of MBL into CLL.

Translational Mini-Review Series on B Cell-Directed Therapies: Recent advances in B cell-directed biological therapies for autoimmune disorders.

B cell-directed therapies are promising treatments for autoimmune disorders. Besides targeting CD20, newer B cell-directed therapies are in development that target other B cell surface molecules and differentiation factors. An increasing number of B cell-directed therapies are in development for the treatment of autoimmune disorders. Like rituximab, which is approved as a treatment for rheumatoid arthritis (RA), many of these newer agents deplete B cells or target pathways essential for B cell development and function; however, many questions remain about their optimal use in the clinic and about the role of B cells in disease pathogenesis. Other therapies besides rituximab that target CD20 are the furthest along in development. Besides targeting CD20, the newer B cell-directed therapies target CD22, CD19, CD40-CD40L, B cell activating factor belonging to the TNF family (BAFF) and A proliferation-inducing ligand (APRIL). Rituximab is being tested in an ever-increasing number of autoimmune disorders and clinical studies of rituximab combined with other biological therapies are being pursued for the treatment of rheumatoid arthritis (RA). B cell-directed therapies are being tested in clinical trials for a variety of autoimmune disorders including RA, systemic lupus erythematosus (SLE), Sjögren's syndrome, vasculitis, multiple sclerosis (MS), Graves' disease, idiopathic thrombocytopenia (ITP), the inflammatory myopathies (dermatomyositis and polymyositis) and the blistering skin diseases pemphigus and bullous pemphigoid. Despite the plethora of clinical studies related to B cell-directed therapies and wealth of new information from these trials, much still remains to be discovered about the pathophysiological role of B cells in autoimmune disorders.

Apolipoprotein E genotype as a determinant of survival in chronic lymphocytic leukemia.

Survival of chronic lymphocytic leukemia (CLL) cells requires sustained activation of the antiapoptotic PI-3-K/Akt pathway, and many therapies for CLL cause leukemia cell death by triggering apoptosis. Blood lipoprotein particles are either pro- or antiapoptotic. High-density lipoprotein particles are antiapoptotic through sphingosine-1-phosphate receptor 3-mediated activation of the PI-3-K/Akt pathway. Apolipoprotein E4 (apoE4)-very low density lipoproteins (VLDL) increase apoptosis, but the apoE2-VLDL and apoE3-VLDL isoforms do not. As increased B-cell apoptosis favors longer survival of CLL patients, we hypothesized that APOE4 genotype would beneficially influence the clinical course of CLL. We report here that women (but not men) with an APOE4 genotype had markedly longer survival than non-APOE4 patients. VLDL is metabolized to low-density lipoprotein through lipoprotein lipase. Higher levels of lipoprotein lipase mRNA in these CLL patients correlated with shorter survival. The beneficial effect of APOE4 in CLL survival is likely mediated through APOE4 allele-specific regulation of leukemia cell apoptosis. The APOE allele and genotype distribution in these CLL patients is the same as in unaffected control populations, suggesting that although APOE genotype influences CLL outcome and response to therapy, it does not alter susceptibility to developing this disease.

Smoking is associated with an age-related decline in exhaled nitric oxide.

Age-related declines in forced expiratory volume in one second are accelerated in smokers. Smoking is associated with decreased exhaled nitric oxide fraction (F(eNO)). The aim of the present study was to determine the impact of age on F(eNO) in otherwise healthy smokers and nonsmokers. F(eNO) and serum cotinine levels were measured in 994 healthy subjects aged 18-40 yrs. American Thoracic Society questionnaire data on smoking habits was used to validate serum cotinine levels as a surrogate marker for categorisation of smokers and nonsmokers in the cohort. Serum cotinine levels were a good discriminator of smokers (n = 99) and nonsmokers (n = 895). F(eNO) levels were significantly lower in otherwise healthy smokers compared with nonsmokers. There was an inverse correlation of serum cotinine levels with F(eNO). No correlation of age with F(eNO) was found in nonsmokers but an inverse correlation of F(eNO) with age in smokers was found. F(eNO) was significantly lower in smokers aged 21-40 yrs compared with nonsmokers aged 21-40 yrs, but was not lower in smokers aged 18-20 yrs compared with nonsmokers of the same age. Smoking was associated with decreased exhaled nitric oxide. The greatest smoking-related declines in exhaled nitric oxide occurred in older subjects. This suggests that smoking is associated with age-related declines in exhaled nitric oxide and justifies future mechanistic studies that address the impact of exhaled nitric oxide decline on lung function.

IL-4 and interferon gamma regulate expression of inducible nitric oxide synthase in chronic lymphocytic leukemia cells.

Chronic lymphocytic leukemia (B-CLL) is characterized by the accumulation of long-lived non-dividing CD5(+) B cells. Nitric oxide (NO) is an important regulator of apoptosis, and the viability of cultured B-CLL cells may be dependent on the autocrine production of nitric oxide by inducible nitric oxide synthase (NOS2). We performed this study to determine whether cytokine factors that prevent spontaneous in vitroapoptosis of B-CLL cells induce B-CLL cell NOS2 enzyme activity. B-CLL cells expressed NOS enzyme activity and NOS2 protein and mRNA. IL-4 and IFN-gamma increased B-CLL cell NOS2 enzyme activity and protein expression during in vitro culture. IFN-gamma, but not IL-4, increased NOS2 mRNA expression in cultured B-CLL cells suggesting that IL-4-mediated changes of NOS2 protein expression occurred at the post-transcriptional level. We were unable to detect increased concentrations of nitrite or nitrate (NO(x)) as surrogate markers of NO production in B-CLL cell cultures treated with IL-4 or IFN-gamma. IL-4 and IFN-gamma diminished NOS inhibitor-induced B-CLL cell death. In summary, we found that B-CLL cells expressed NOS2 and that IL-4 and IFN-gamma increased B-CLL NOS2 expression. Cytokine-mediated expression of NOS2 by B-CLL cells may promote their survival, and therapeutic strategies that target NOS2 or quench NO may be beneficial in patients with B-CLL.

Nitric oxide enhancement of fludarabine cytotoxicity for B-CLL lymphocytes.

Fludarabine is active but not curative in the treatment of chronic lymphocytic leukemia (B-CLL). Nitric oxide (NO) supplied from exogenous, NO-donating pro-drugs can also induce apoptosis and death of acute leukemia cells. This study investigated combinations of fludarabine with NO-donating pro-drugs for their cytotoxicity against freshly isolated B-CLL lymphocytes following a 72 h exposure in vitro. The median IC(50)for fludarabine was 2.2 microM (n = 85). The nitric oxide donors DETA-NO, PAPA-NO, and MAHMA-NO were also cytotoxic, and their effects were inversely related to rates of NO release. Neither DETA-NO depleted of NO nor DETA itself was effective, indicating that NO was required for cytotoxicity. Drug interactions were evaluated by a modified combination index method. Synergy was observed in combinations of fludarabine or nelarabine (506U78) with DETA-NO in 52% and 88% of samples, respectively. Interestingly, the combination of fludarabine and DETA-NO was more cytotoxic in B-CLL cells less sensitive to fludarabine. DETA-NO did not enhance the activity of other DNA anti-metabolites, topoisomerase I and II inhibitors, or alkylating agents. Finally, the anti-leukemic activity of fludarabine alone or in combination with DETA-NO was found to correlate with inhibition of cellular RNA synthesis. These results indicate that NO donors could enhance fludarabine therapy for B-CLL.

Activated T lymphocytes regulate hyaluronan binding to monocyte CD44 via production of IL-2 and IFN-gamma.

Interactions of the cell surface proteoglycan CD44 with the extracellular matrix glycosaminoglycan hyaluronan (HA) are important during inflammatory immune responses. Our previous studies indicated that monocyte HA binding could be induced by TNF-alpha. Moreover, monocyte HA binding could be markedly up-regulated by culturing PBMC with anti-CD3 (TCR complex) mAbs. The present study was undertaken to identify soluble factors and/or cell surface molecules of activated T lymphocytes that might regulate HA binding to monocytes. Abs to IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-10, IL-15, GM-CSF, IFN-gamma, and TNF-alpha were tested for their effects on anti-CD3 mAb-, Con A-, and PMA/ionomycin-mediated monocyte HA binding in PBMC cultures. Anti-TNF-alpha, anti-IL-2, and anti-IFN-gamma Abs, when added together to PBMC cultures, completely blocked Con A- and partially blocked anti-CD3- and PMA/ionomycin-induced monocyte HA binding. Furthermore, when added together to PBMC cultures, IL-2 and TNF-alpha induced high levels of monocyte HA binding. Likewise, IFN-gamma augmented TNF-alpha-induced monocyte HA binding. To investigate the role of T cell-monocyte direct contact in induction of monocyte HA binding, we studied PMA/ionomycin-activated, paraformaldehyde-fixed CD4(+) T cells in these assays. Fixed, PMA/ionomycin-activated CD4(+) T lymphocytes induced monocyte HA binding, but direct T cell-monocyte contact was not required. Moreover, anti-IFN-gamma and anti-TNF-alpha Abs blocked fixed PMA/ionomycin-activated CD4(+) T cell-induced monocyte HA binding. Taken together, these studies indicate roles for soluble T lymphocyte-derived factor(s), such as IL-2 and IFN-gamma, and a role for monocyte-derived TNF-alpha in Con A-, TCR complex-, and PMA/ionomycin-induced HA binding to monocyte CD44.

Serum levels of soluble CD44 in primary Sjögren's syndrome.

OBJECTIVE: To determine whether elevated soluble CD44 (sCD44) levels serve as a marker of inflammation and lymphoproliferation in primary Sjögren's syndrome (SS). METHODS: We measured sCD44 levels by ELISA in serum samples from a cross section of healthy individuals and patients seen in a rheumatology clinic for evaluation of possible primary SS. RESULTS: Median serum levels of sCD44 were significantly higher in 48 healthy men compared to 52 healthy women (16 vs. 12 nmol/l; p = 0.0034). There was no relationship between serum levels of sCD44 and age or ethnic background. Slightly higher median levels of sCD44 were found in the serum of 37 women with primary SS compared to healthy women (14 vs. 12 nmol/l; p = 0.0402). However, these levels were comparable to those of 33 female patients without primary SS who were seen in the same clinic (p = 0.1233). CONCLUSION: Serum levels of sCD44 were slightly higher in female patients with primary SS compared to healthy women, but they are not likely to discriminate between patients with and without primary SS in a rheumatology practice.

Nitric oxide synthase type 2 promoter polymorphisms, nitric oxide production, and disease severity in Tanzanian children with malaria.

Nitric oxide (NO) plays an important role in host resistance to infection with a variety of organisms. Two recent reports from Gabon and Gambia identified associations of malaria disease severity with the inducible NO synthase (NOS2) promoter G-954C and short allele (<11 repeats) pentanucleotide microsatellite polymorphisms, respectively. It was postulated that there would be a correlation of these polymorphisms with malaria disease severity and with measures of NO production in our cohort of Tanzanian children with malaria. In Tanzanian children, 15% were heterozygous or homozygous for the G-954C polymorphism, and 13% had the short-allele microsatellite polymorphism. There was no significant correlation of either polymorphism with disease severity or with measures of NO production and NOS2 expression. Black and white Americans differed significantly in the frequencies of these polymorphisms. The various association of these gene polymorphisms with malaria severity in different populations underscores the complexity of host resistance to malaria.

TNFalpha and IL-4 regulation of hyaluronan binding to monocyte CD44 involves posttranslational modification of CD44.

Our previous studies have identified TNFalpha as a positive regulator and IL-4 as a negative regulator of human monocyte CD44-HA binding. In order to determine the mechanisms of IL-4- and TNFalpha-mediated regulation of monocyte HA binding, we measured HA binding and CD44 expression on peripheral blood monocytes following monocyte treatment with TNFalpha or IL-4, as well as following monocyte treatment with inhibitors of protein synthesis, N- and O-linked glycosylation, and chondroitin sulfation. IL-4 decreased CD44-HA binding on monocytes initially treated with TNFalpha. Similarly, pretreatment of monocytes with IL-4 prevented subsequent TNFalpha-mediated HA binding. Cycloheximide (protein synthesis inhibitor), tunicamycin (N-linked glycosylation inhibitor), and beta-d-xyloside (chondroitin sulfation inhibitor) all inhibited IL-4-mediated downregulation of TNFalpha-induced monocyte HA binding. Western blot analysis of CD44 from TNFalpha-treated monocytes revealed a 5-10 Mr decrease in the standard isoform of CD44. In contrast, IL-4 treatment of monocytes inhibited CD44-HA binding and reversed the 5- to 10-kDa decrease in monocyte CD44 Mr. Finally, studies with F10.44.2, a CD44 mab that enhances CD44-HA binding, indicated that IL-4 treatment of monocytes not only diminished constitutive HA binding, but also diminished CD44 mab-induced HA binding. Taken together, these data suggested that IL-4-mediated inhibition of TNFalpha-induced monocyte HA binding was dependent not only on protein synthesis, but also on N-linked glycosylation and chondroitin-sulfate modification of either CD44 or, alternatively, another monocyte protein(s) that may regulate the ability of CD44 to bind HA.

Interferon-beta1A-induced polyarthritis in a patient with the HLA-DRB1*0404 allele.

Human interferon-alpha (IFNalpha) and IFNbeta are administered for treatment of several diseases, including viral infections, malignancies, and multiple sclerosis (MS). IFNalpha therapy has been associated with the production of autoantibodies and the development of a variety of autoimmune disorders, including polyarthritis. This report describes the development of seronegative, symmetric polyarthritis in a patient with relapsing-remitting MS, after 8 weeks of therapy with IFNbeta1a. HLA phenotyping analysis of the patient revealed the presence of HLA-DRB1*0404, an allele known to be associated with the development of rheumatoid arthritis. Therefore, IFNbeta1a may have induced arthritis in a patient who was genetically predisposed to develop arthritis on the basis of HLA determinants. The English-language literature regarding IFNalpha- and IFNbeta-induced polyarthritis is reviewed, and possible mechanisms for IFNalpha- and IFNbeta-induced autoimmunity, including the contribution of HLA determinants and nitric oxide overproduction, are discussed.

Cytokine-regulated expression of activated leukocyte cell adhesion molecule (CD166) on monocyte-lineage cells and in rheumatoid arthritis synovium.

OBJECTIVE: To determine whether monocyte/macrophage expression of the CD6 ligand, activated leukocyte cell adhesion molecule (ALCAM) (CD166), is regulated by cytokines during inflammation in rheumatoid arthritis (RA). METHODS: We used flow cytometry to test whether cytokines present in rheumatoid synovium could regulate ALCAM cell surface expression on peripheral blood (PB) monocytes and RA synovial fluid (SF) macrophages, and we examined ALCAM expression in situ in RA synovium by immunofluorescence. RESULTS: The monocyte differentiation factors interleukin-3, macrophage colony-stimulating factor (M-CSF), and granulocyte-macrophage colony-stimulating factor augmented ALCAM expression on PB monocytes. ALCAM was expressed on monocyte-lineage cells in situ in inflamed synovium from patients with RA (9 of 9), but not in uninflamed synovium from patients with joint trauma (0 of 3). Furthermore, in vitro culture-induced ALCAM expression on PB monocytes and CD14+ RA SF cells was inhibited by an M-CSF neutralizing antibody. CONCLUSION: ALCAM expression on PB and SF monocytes/macrophages is enhanced by M-CSF.

Recombinant acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) purified to essential homogeneity utilizes cholesterol in mixed micelles or in vesicles in a highly cooperative manner.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl coenzyme A. The first gene encoding the enzyme, designated as ACAT-1, was identified in 1993 through an expression cloning approach. We isolated a Chinese hamster ovary cell line that stably expresses the recombinant human ACAT-1 protein bearing an N-terminal hexahistidine tag. We purified this enzyme approximately 7000-fold from crude cell extracts by first solubilizing the cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, then proceeding with an ACAT-1 monoclonal antibody affinity column and an immobilized metal affinity column. The final preparation is enzymologically active and migrates as a single band at 54 kDa on SDS-polyacrylamide gel electrophoresis. Pure ACAT-1 dispersed in mixed micelles containing sodium taurocholate, phosphatidylcholine, and cholesterol remains catalytically active. The cholesterol substrate saturation curves of the enzyme assayed either in mixed micelles or in reconstituted vesicles are both highly sigmoidal. The oleoyl-coenzyme A substrate saturation curves of the enzyme assayed under the same conditions are both hyperbolic. These results support the hypothesis that ACAT is an allosteric enzyme regulated by cholesterol.

Expression of CD44 and variant isoforms in cultured human retinal pigment epithelial cells.

PURPOSE: CD44 is a major hyaluronic acid receptor that exists as a number of isoforms, generated by alternative splicing of 9 "variant" exons in humans (v2 to v10) and 10 exons in rodents. Little is known about the expression and function of CD44 in human retinal pigment epithelium (RPE) cells. Therefore, the authors determined whether human RPE cells express CD44, and whether the expression differs depending on the proliferative status of the cells. METHODS: Human RPE cells were harvested from normal donor eyes and propagated in culture. Total RNA was extracted from cultured cells. mRNA expression of CD44 was determined by reverse transcription-polymerase chain reaction (RT-PCR), followed by cloning and sequencing of the PCR products, and by Southern hybridization. CD44 cell surface expression was measured by flow cytometry. Western hybridization and immunohistochemistry were used to determine the CD44 immunoreactivity of cultured human RPE cells and normal human RPE cells in situ. RESULTS: The standard form of CD44 mRNA and variant isoforms containing exon v6 or v10 were expressed in cultured human RPE cells. CD44 mRNA and protein levels were increased in proliferating human RPE cells compared with density-arrested counterparts. Addition of 1 microM retinoic acid enhanced the cell density-induced downregulation of CD44 mRNA, but did not significantly affect the CD44 cell surface protein expression. As previously reported, CD44 immunoreactivity was not detected in normal human RPE cells in situ. CONCLUSIONS: Cultured human RPE cells express CD44 standard form and variant isoforms containing exon v6 or v10, which are preferentially expressed by proliferating human RPE cells.

Cytokine induction of the ability of human monocyte CD44 to bind hyaluronan is mediated primarily by TNF-alpha and is inhibited by IL-4 and IL-13.

Ligation of CD44 by hyaluronan (HA) is a key proinflammatory event that regulates lymphocyte and monocyte adhesion and cytokine production. While most immune cells express CD44, few immune cells constitutively bind HA. We have previously shown that monocyte CD44 acquires the ability to bind HA after in vitro culture of PBMC in human serum and, therefore, we have investigated a series of human cytokines and bacterial LPS for their ability to induce and/or inhibit monocyte CD44 to bind HA. We found that IL-1alpha, IL-1beta, IL-3, granulocyte macrophage (GM)-CSF, and TNF-alpha, as well as bacterial LPS, all directly induced peripheral blood monocytes to bind HA. In contrast, IL-2 and IL-15 up-regulated monocyte CD44 HA-binding in PBMC suspensions, but not in purified monocyte suspensions. An anti-TNF-alpha-neutralizing Ab inhibited IL-1alpha-, IL-1beta-, IL-3-, and GM-CSF-mediated monocyte HA binding. In addition, treatment of IL-2- and IL-15-stimulated PBMC cultures with an anti-TNF-alpha Ab prevented IL-2- and IL-15-induced monocyte HA binding, thus identifying TNF-alpha as a lymphocyte-derived factor that acted on monocytes to induce HA binding. In contrast, IL-4 and IL-13 were potent inhibitors of monocyte CD44-HA binding induced by either human serum or by IL-1alpha, IL-1beta, IL-3, GM-CSF, or TNF-alpha. IL-10 had dual effects on monocyte CD44-HA binding. Alone, IL-10 induced HA binding to PBMC monocyte CD44, while in contrast, IL-10 inhibited IL-1-induced monocyte CD44 binding to HA. Taken together, these studies identify a network of T cell and monocyte-derived cytokines that regulate HA binding to peripheral blood monocyte CD44, primarily through TNF-alpha.