Brain activity during Stroop task performance at age 74 after exposure to the Dutch famine during early gestation.

OBJECTIVE: Poorer performance on the Stroop task has been reported after prenatal famine exposure at age 58, potentially indicating cognitive decline. We investigated whether brain activation during Stroop task performance at age 74 differed between individuals exposed to famine prenatally, individuals born before and individuals conceived after the famine. METHOD: In the Dutch famine birth cohort, we performed a Stroop task fMRI study of individuals exposed (n = 22) or unexposed (born before (n = 18) or conceived after (n = 25)) to famine in early gestation. We studied group differences in task-related mean activation of the dorsolateral prefrontal cortex (DLPFC), anterior cingulate cortex (ACC) and posterior parietal cortex (PPC). Additionally, we explored potential disconnectivity of the DLPFC using psychophysiological interaction analysis. RESULTS: We observed similar activation patterns in the DLPFC, ACC and PPC in individuals born before and individuals exposed to famine, while individuals conceived after famine had generally higher activation patterns. However, activation patterns were not significantly different between groups. Task-related decreases in connectivity were observed between left DLPFC-left PPC and right DLPFC-right PPC, but were not significantly different between groups. CONCLUSIONS: Although not statistically significant, the observed patterns of activation may reflect a combined effect of general brain aging and prenatal famine exposure.

Differences in Treatment Response in Bronchial Epithelial Cells from Idiopathic Pulmonary Fibrosis (IPF) Patients: A First Step towards Personalized Medicine?

Idiopathic pulmonary fibrosis (IPF) has a detrimental prognosis despite antifibrotic therapies to which individual responses vary. IPF pathology is associated with oxidative stress, inflammation and increased activation of SRC family kinases (SFK). This pilot study evaluates individual responses to pirfenidone, nintedanib and SFK inhibitor saracatinib, markers of redox homeostasis, fibrosis and inflammation, in IPF-derived human bronchial epithelial (HBE) cells. Differentiated HBE cells from patients with and without IPF were analyzed for potential alterations in redox and profibrotic genes and pro-inflammatory cytokine secretion. Additionally, the effects of pirfenidone, nintedanib and saracatinib on these markers were determined. HBE cells were differentiated into a bronchial epithelium containing ciliated epithelial, basal, goblet and club cells. NOX4 expression was increased in IPF-derived HBE cells but differed on an individual level. In patients with higher NOX4 expression, pirfenidone induced antioxidant gene expression. All drugs significantly decreased NOX4 expression. IL-6 (p = 0.09) and IL-8 secretion (p = 0.014) were increased in IPF-derived HBE cells and significantly reduced by saracatinib. Finally, saracatinib significantly decreased TGF-ß gene expression. Our results indicate that treatment responsiveness varies between IPF patients in relation to their oxidative and inflammatory status. Interestingly, saracatinib tends to be more effective in IPF than standard antifibrotic drugs.

Shaping the risk for late-life neurodegenerative disease: A systematic review on prenatal risk factors for Alzheimer's disease-related volumetric brain biomarkers.

Environmental exposures including toxins and nutrition may hamper the developing brain in utero, limiting the brain's reserve capacity and increasing the risk for Alzheimer's disease (AD). The purpose of this systematic review is to summarize all currently available evidence for the association between prenatal exposures and AD-related volumetric brain biomarkers. We systematically searched MEDLINE and Embase for studies in humans reporting on associations between prenatal exposure(s) and AD-related volumetric brain biomarkers, including whole brain volume (WBV), hippocampal volume (HV) and/or temporal lobe volume (TLV) measured with structural magnetic resonance imaging (PROSPERO; CRD42020169317). Risk of bias was assessed using the Newcastle Ottawa Scale. We identified 79 eligible studies (search date: August 30th, 2020; Ntotal=24,784; median age 10.7 years) reporting on WBV (N = 38), HV (N = 63) and/or TLV (N = 5) in exposure categories alcohol (N = 30), smoking (N = 7), illicit drugs (N = 14), mental health problems (N = 7), diet (N = 8), disease, treatment and physiology (N = 10), infections (N = 6) and environmental exposures (N = 3). Overall risk of bias was low. Prenatal exposure to alcohol, opioids, cocaine, nutrient shortage, placental dysfunction and maternal anemia was associated with smaller brain volumes. We conclude that the prenatal environment is important in shaping the risk for late-life neurodegenerative disease.

Impaired JAK-STAT pathway signaling in leukocytes of the frail elderly.

BACKGROUND: Elderly often show reduced immune functioning and can develop chronic low-grade inflammation. Why some elderly are more prone to become frail is unknown. We investigated whether frailty is associated with altered cytokine signaling through the JAK-STAT pathway in leukocytes of 34 individuals aged 65-74 years. In addition, we investigated how this relation is affected by chronic low-grade inflammation during the previous 20 years. Cytokine signaling was quantified by measuring intracellular STAT1, STAT3, and STAT5 phosphorylation in monocytes, B cells, CD4+ T cells and CD8+ T cells upon stimulation with IL-2, IL-6, IL-10, IFNα and IFNγ, using phospho-flow cytometry. Presence of chronic low-grade inflammation was investigated by evaluating 18 different plasma inflammatory markers that had been measured repeatedly in the same individuals over the previous 20 years. Frailty was assessed as a score on a frailty index. RESULTS: We found that lower cytokine-induced pSTAT responsiveness in the various cell subsets was seen with higher frailty scores in both men and women, indicative of dysfunctional pSTAT responses in frailer individuals. Associations differed between men and women, with frailer women showing lower pSTAT1 responses in monocytes and frailer men showing lower pSTAT5 responses in CD4+ and CD8+ T cells. Notably, lower IL-10-induced pSTAT3 responses in men were related to both higher frailty scores and higher CRP levels over the past 20 years. This might indicate poor resolution of low-grade inflammation due to defective regulatory pSTAT signaling in older men. CONCLUSIONS: Our results emphasize the importance of preserved JAK-STAT pathway signaling in healthy aging and reveal cellular pSTAT levels as a candidate biomarker of frailty.

The use of exhaled air analysis in discriminating interstitial lung diseases: a pilot study.

BACKGROUND: Fibrotic Interstitial lung diseases (ILD) are a heterogeneous group of chronic lung diseases characterized by diverse degrees of lung inflammation and remodeling. They include idiopathic ILD such as idiopathic pulmonary fibrosis (IPF), and ILD secondary to chronic inflammatory diseases such as connective tissue disease (CTD). Precise differential diagnosis of ILD is critical since anti-inflammatory and immunosuppressive drugs, which are beneficial in inflammatory ILD, are detrimental in IPF. However, differential diagnosis of ILD is still difficult and often requires an invasive lung biopsy. The primary aim of this study is to identify volatile organic compounds (VOCs) patterns in exhaled air to non-invasively discriminate IPF and CTD-ILD. As secondary aim, the association between the IPF and CTD-ILD discriminating VOC patterns and functional impairment is investigated. METHODS: Fifty-three IPF patients, 53 CTD-ILD patients and 51 controls donated exhaled air, which was analyzed for its VOC content using gas chromatograph- time of flight- mass spectrometry. RESULTS: By applying multivariate analysis, a discriminative profile of 34 VOCs was observed to discriminate between IPF patients and healthy controls whereas 11 VOCs were able to distinguish between CTD-ILD patients and healthy controls. The separation between IPF and CTD-ILD could be made using 16 discriminating VOCs, that also displayed a significant correlation with total lung capacity and the 6 min' walk distance. CONCLUSIONS: This study reports for the first time that specific VOC profiles can be found to differentiate IPF and CTD-ILD from both healthy controls and each other. Moreover, an ILD-specific VOC profile was strongly correlated with functional parameters. Future research applying larger cohorts of patients suffering from a larger variety of ILDs should confirm the potential use of breathomics to facilitate fast, non-invasive and proper differential diagnosis of specific ILDs in the future as first step towards personalized medicine for these complex diseases.

In-depth immune cellular profiling reveals sex-specific associations with frailty.

BACKGROUND: With advancing age, the composition of leukocyte subpopulations in peripheral blood is known to change, but how this change differs between men and women and how it relates to frailty is poorly understood. Our aim in this exploratory study was to investigate whether frailty is associated with changes in immune cell subpopulations and whether this differs between men and women. Therefore, we performed in-depth immune cellular profiling by enumerating a total of 37 subpopulations of T cells, B cells, NK cells, monocytes, and neutrophils in peripheral blood of 289 elderly people between 60-87 years of age. Associations between frailty and each immune cell subpopulation were tested separately in men and women and were adjusted for age and CMV serostatus. In addition, a random forest algorithm was used to predict a participant's frailty score based on enumeration of immune cell subpopulations. RESULTS: In the association study, frailty was found to be associated with increased numbers of neutrophils in both men and in women. Frailer women, but not men, showed higher numbers of total and CD16- monocytes, and lower numbers of both CD56+ T cells and late differentiated CD4+ TemRA cells. The random forest algorithm confirmed all the findings of the association studies in men and women. In men, the predictive accuracy of the algorithm was too low (5.5%) to warrant additional conclusions on top of the ones derived from the association study. In women however, the predictive accuracy was higher (23.1%), additionally revealing that total T cell numbers and total lymphocyte numbers also contribute in predicting frailty. CONCLUSIONS: In-depth immune cellular profiling revealed consistent associations of frailty with elevated numbers of myeloid cell subpopulations in both men and women. Furthermore, additional associations were found between frailty and lower numbers of some T cell subpopulations, in women only. Thus, our study indicates sex-specific associations of immune subpopulations with frailty. We hope that our study will prompt further investigation into the sex-specific immune mechanisms associated with the development of frailty.

Frailty is associated with elevated CRP trajectories and higher numbers of neutrophils and monocytes.

BACKGROUND: With aging, the human immune system undergoes several changes. The clinical relevance of these changes, however, is relatively unknown. We investigated immunological aspects of human aging in relation to frailty in the Doetinchem Cohort Study (DCS). METHODS: We calculated a frailty index score based on 36 health parameters for each individual in the DCS with data obtained in the period 2008-2016. The frailty index was used to define three health groups ('healthy', 'intermediate', and 'frail'), stratified by age and sex. In a subcohort (n = 289, 60-85 years, selected by balanced random sampling per frailty group), we collected blood samples between October 2016 and March 2017 to determine absolute numbers of leukocyte subsets. In addition, cytomegalovirus serostatus was assessed. C-reactive protein (CRP) levels were longitudinally assessed in four consecutive plasma samples per individual. These samples had been previously collected (1993-2013) as part of the DCS at regular time intervals and spanning a period of >15 years. RESULTS: We observed higher numbers of myeloid derived neutrophils and monocytes in the frail group compared to the healthy group in both men and women, and, retrospectively, consistently higher CRP concentrations over a period of >15 years. An increase in CRP concentration with age was found in women, but not in men. Frailty was not associated with cytomegalovirus serostatus or with changes in lymphoid derived T-, B-, or NK-cell numbers. CONCLUSION: Frail elderly, compared to their age- and sex-matched peers, endure a chronic and stable low-grade inflammation, which is associated with a myeloid cell lineage expansion. These findings could help to monitor clinically significant immunological decline in the elderly.

Exposure to genotoxic compounds alters in vitro cellular VOC excretion.

Genotoxic carcinogens significantly damage cells and tissues by targeting macromolecules such as proteins and DNA, but their mechanisms of action and effects on human health are diverse. Consequently, determining the amount of exposure to a carcinogen and its cellular effects is essential, yet difficult. The aim of this manuscript was to investigate the potential of detecting alterations in volatile organic compounds (VOCs) profiles in the in vitro headspace of pulmonary cells after exposure to the genotoxic carcinogens cisplatin and benzo[a]pyrene using two different sampling set-ups. A prototype set-up was used for the cisplatin exposure, whereas a modified set-up was utilized for the benzo[a]pyrene exposure. Both carcinogens were added to the cell medium for 24 h. The headspace in the culture flask was sampled to measure the VOC content using gas chromatography-time-of-flight-mass spectrometry. Eight cisplatin-specific VOCs and six benzo[a]pyrene-specific VOCs were discriminatory between treated and non-treated cells. Since the in vivo biological effects of both genotoxic compounds are well-defined, the origin of the identified VOCs could potentially be traced back to common cellular processes including cell cycle pathways, DNA damage and repair. These results indicate that exposing lung cells to genotoxins alters headspace VOC profiles, suggesting that it might be possible to monitor VOC changes in vivo to study drug efficacy or exposure to different pollutants. In conclusion, this study emphasizes the innovative potential of in vitro VOCs experiments to determine their in vivo applicability and discover their endogenous origin.

The disturbed redox-balance in pulmonary fibrosis is modulated by the plant flavonoid quercetin.

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed pulmonary redox balance associated with inflammation. To restore this balance, antioxidants are often suggested as therapy for IPF but previous clinical trials with these compounds and their precursors have not been successful in the clinic. The exogenous antioxidant quercetin, which has a versatile antioxidant profile and is effective in restoring a disturbed redox balance, might be a better candidate. The aim of this study was to evaluate the protective effect of quercetin on oxidative and inflammatory markers in IPF. Here, we demonstrate that IPF patients have a significantly reduced endogenous antioxidant defense, shown by a reduced total antioxidant capacity and lowered glutathione and uric acid levels compared to healthy controls. This confirms that the redox balance is disturbed in IPF. Ex vivo incubation with quercetin in blood of both IPF patients and healthy controls reduces LPS-induced production of the pro-inflammatory cytokines IL-8 and TNFα. This anti-inflammatory effect was more pronounced in the blood of the patients. Our pro-fibrotic in vitro model, consisting of bleomycin-triggered BEAS-2B cells, shows that quercetin boosts the antioxidant response, by increasing Nrf2 activity, and decreases pro-inflammatory cytokine production in a concentration-dependent manner. Collectively, our findings implicate that IPF patients may benefit from the use of quercetin to restore the disturbed redox balance and reduce inflammation.

Effect of interleukin (IL)-8 on benzo[a]pyrene metabolism and DNA damage in human lung epithelial cells.

It has been well established that inflammation and concurrent mutagenic exposures drive the carcinogenic process in a synergistic way. To elucidate the role of the inflammatory cytokine IL-8 in this process, we studied its effect on the activation and deactivation of the chemical mutagen benzo[a]pyrene B[a]P in the immortalized pulmonary BEAS-2B cell line. After 24h incubation with B[a]P in the presence or absence of IL-8, the B[a]P induced cytochrome P450 1A1 and 1B1 (CYP1A1 and CYP1B1) gene expression and CYP1A1 enzyme activity was significantly higher in the presence of the cytokine. Consistent with these findings, we observed higher concentration of the metabolite B[a]P-7,8-diol under concurrent IL-8 treatment conditions. Interestingly, we also found higher concentrations of unmetabolized B[a]P. To explain this, we examined the downstream effects of IL-8 on NADPH oxidases (NOXes). IL-8 lowered the intracellular NADPH level, but this effect could not explain the changes in B[a]P metabolism. IL-8 also significantly depleted intracellular glutathione (GSH), which also resulted in enhanced levels of unmetabolized B[a]P, but increased concentrations of the metabolite B[a]P-7,8-diol. No differences in B[a]P-DNA adducts level were found between B[a]P and B[a]P combined with IL-8, and this might be due to a 3-fold increase in nucleotide excision repair (NER) after IL-8 treatment. These findings suggest that IL-8 increased the formation of B[a]P-7,8-diol despite an overall delayed B[a]P metabolism via depletion of GSH, but DNA damage levels were unaffected due to an increase in NER capacity.

Dynamic collection and analysis of volatile organic compounds from the headspace of cell cultures.

Exhaled breath has proven to be a valuable source of information about human bodies. Subtle differences between volatile organic compounds (VOCs) formed endogenously can be detected and become a base for a potential monitoring tool for health and disease. Until now, there has been a lack of biological and mechanistic knowledge of the processes involved in the production of relevant VOCs. Among the possible sources of health-related and disease-related VOCs are microorganisms found in the respiratory tract and in the gut. Other VOCs in the body are produced by cells that are influenced by the disease, for instance, due to metabolic disorders and/or inflammation. To gain insight into the in vivo production of VOCs by human cells and thus the exhaled breath composition, in vitro experiments involving relevant cells should be studied because they may provide valuable information on the production of VOCs by the affected cells. To this aim we developed and validated a system for dynamically (continuously) collecting headspace air in vitro using a Caco-2 cell line. The system allows the application of different cell lines as well as different experimental setups, including varying exposure times and treatment options while preserving cell viability. Significant correlation (p ⩽ 0.0001) between collection outputs within each studied group confirmed high reproducibility of the collection system. An example of such an application is presented here. We studied the influence of oxidative stress on the VOC composition of the headspace air of Caco-2 cells. By comparing the VOC composition of air flushed through empty culture flasks (n = 35), flasks with culture medium (n = 35), flasks with medium and cells (n = 20), flasks with medium and an oxidative stressor (H2O2) (n = 20), and flasks with medium, stressor, and cells (n = 20), we were able to separate the effects from the stressor on the cells from all other interactions. Measurements were performed with gas chromatography time-of-flight mass spectrometry. Multivariate data analysis allowed detection of significant altered compounds in the compared groups. We found a significant change (p ⩽ 0.001) of the composition of VOCs due to the stressing of the Caco-2 cells by H2O2. A total of ten VOCs showed either increased or decreased abundance in the headspace of the cell cultures due to the presence of the H2O2 stressor.

Identification of microorganisms based on headspace analysis of volatile organic compounds by gas chromatography-mass spectrometry.

The identification of specific volatile organic compounds (VOCs) produced by microorganisms may assist in developing a fast and accurate methodology for the determination of pulmonary bacterial infections in exhaled air. As a first step, pulmonary bacteria were cultured and their headspace analyzed for the total amount of excreted VOCs to select those compounds which are exclusively associated with specific microorganisms. Development of a rapid, noninvasive methodology for identification of bacterial species may improve diagnostics and antibiotic therapy, ultimately leading to controlling the antibiotic resistance problem. Two hundred bacterial headspace samples from four different microorganisms (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Klebsiella pneumoniae) were analyzed by gas chromatography-mass spectrometry to detect a wide array of VOCs. Statistical analysis of these volatiles enabled the characterization of specific VOC profiles indicative for each microorganism. Differences in VOC abundance between the bacterial types were determined using ANalysis of VAriance-principal component analysis (ANOVA-PCA). These differences were visualized with PCA. Cross validation was applied to validate the results. We identified a large number of different compounds in the various headspaces, thus demonstrating a highly significant difference in VOC occurrence of bacterial cultures compared to the medium and between the cultures themselves. Additionally, a separation between a methicillin-resistant and a methicillin-sensitive isolate of S. aureus could be made due to significant differences between compounds. ANOVA-PCA analysis showed that 25 VOCs were differently profiled across the various microorganisms, whereas a PCA score plot enabled the visualization of these clear differences between the bacterial types. We demonstrated that identification of the studied microorganisms, including an antibiotic susceptible and resistant S. aureus substrain, is possible based on a selected number of compounds measured in the headspace of these cultures. These in vitro results may translate into a breath analysis approach that has the potential to be used as a diagnostic tool in medical microbiology.

Apoptotic, inflammatory, and fibrogenic effects of two different types of multi-walled carbon nanotubes in mouse lung.

There is increasing concern about the toxicity of inhaled multi-walled carbon nanotubes (MWCNTs). Pulmonary macrophages represent the primary cell type involved in the clearance of inhaled particulate materials, and induction of apoptosis in these cells has been considered to contribute to the development of lung fibrosis. We have investigated the apoptotic, inflammogenic, and fibrogenic potential of two types of MWCNTs, characterised by a contrasting average tube length and entanglement/agglomeration. Both nanotube types triggered H2O2 formation by RAW 264.7 macrophages, but in vitro toxicity was exclusively seen with the longer MWCNT. Both types of nanotubes caused granuloma in the mouse lungs. However, the long MWCNT induced a more pronounced pro-fibrotic (mRNA expression of matrix metalloproteinase-8 and tissue inhibitor of metalloproteinase-1) and inflammatory (serum level of monocyte chemotactic protein-1) response. Masson trichrome staining also revealed epithelial cell hyperplasia for this type of MWCNT. Enhanced apoptosis was detected by cleaved caspase 3 immunohistochemistry in lungs of mice treated with the long and rigid MWCNT and, to a lesser extent, with the shorter, highly agglomerated MWCNT. However, staining was merely localised to granulomatous foci, and neither of the MWCNTs induced apoptosis in vitro, evaluated by caspase 3/7 activity in RAW 264.7 cells. In addition, our study reveals that the inflammatory and pro-fibrotic effects of MWCNTs in the mouse lung can vary considerably depending on their composition. The in vitro analysis of macrophage apoptosis appears to be a poor predictor of their pulmonary hazard.

Comprehensive analysis of miRNA expression in T-cell subsets of rheumatoid arthritis patients reveals defined signatures of naive and memory Tregs.

Disturbed expression of microRNAs (miRNAs) in regulatory T cells (Tregs) leads to development of autoimmunity in experimental mouse models. However, the miRNA expression signature characterizing Tregs of autoimmune diseases, such as rheumatoid arthritis (RA) has not been determined yet. In this study, we have used a microarray approach to comprehensively analyze miRNA expression signatures of both naive Tregs (CD4+CD45RO-CD25++) and memory Tregs (CD4+CD45RO+CD25+++), as well as conventional naive (CD4+CD45RO-CD25-) and memory (CD4+CD45RO+CD25-) T cells (Tconvs) derived from peripheral blood of RA patients and matched healthy controls. Differential expression of selected miRNAs was validated by TaqMan-based quantitative reverse transcription-PCR. We found a positive correlation between increased expression of miR-451 in T cells of RA patients and disease activity score (DAS28), erythrocyte sedimentation rate levels and serum levels of interleukin-6. Moreover, we found characteristic, disease- and treatment-independent, global miRNA expression signatures defining naive Tregs, memory Tregs, naive Tconvs and memory Tconvs. The analysis allowed us to define miRNAs characteristic for a general naive phenotype (for example, miR-92a) and a general memory phenotype (for example, miR-21, miR-155). Importantly, the analysis allowed us to define miRNAs that are specifically expressed in both naive and memory Tregs, defining as such miRNA signature characterizing the Treg phenotype (that is, miR-146a, miR-3162, miR-1202, miR-1246 and miR-4281).

Therapeutic dosing of an orally active, selective cathepsin S inhibitor suppresses disease in models of autoimmunity.

The purpose of the study was to examine the potential of inhibition of cathepsin S as a treatment for autoimmune diseases. A highly selective cathepsin S inhibitor, CSI-75, was shown to upregulate levels of the cathepsin S substrate, invariant chain Lip10, in vitro as well as in vivo in C57Bl/6 mice after oral administration. Functional activity of the compound was shown by a reduction in the OVA-specific response of OVA-sensitized splenocytes from C57Bl/6 mice as well as from OVA-TCR transgenic mice (DO11.10). Since these studies revealed a selective suppression of the Th1 and Th17 cytokines causing a shift to Th2, CSI-75 was tested in the murine HC-gp39-immunization model. Indeed, CSI-75 specifically reduced the circulating HC-gp39-specific IgG2a in these mice indicating selective inhibition of the Th1 type of response in vivo. The importance of especially the Th1 and Th17 cell subsets in the pathology of autoimmune diseases, renders CatS inhibition a highly interesting potential therapeutic treatment of autoimmune diseases. Therefore, CSI-75 was tested in a murine model of multiple sclerosis (i.e. experimental autoimmune encephalomyelitis (EAE)) in a semi-therapeutic setting (ie. oral treatment after initial sensitization to antigen). Finally, in a murine model with features resembling rheumatoid arthritis (the collagen-induced arthritis (CIA) model), CSI-75 was tested in a therapeutic manner (after disease development). CSI-75 caused a significant reduction in disease score in both disease models, indicating a promising role for CatS inhibitors in the area of therapeutic treatments for autoimmune diseases.

Mycophenolic acid-mediated suppression of human CD4+ T cells: more than mere guanine nucleotide deprivation.

Mycophenolic acid is the active ingredient of the immunosuppressant mycophenolate mofetil that is widely used in transplantation medicine and autoimmunity. Mycophenolic acid inhibits inosine monophosphate dehydrogenase, an enzyme involved in biosynthesis of guanine nucleotides required for lymphocyte clonal expansion. Here, we present novel insights into the mechanisms underlying mycophenolic acid-mediated suppression of human CD4+ T cells. Upon CD3/CD28 stimulation, mycophenolic acid inhibited T cell IL-17, IFN-γ and TNF-α production but not IL-2 production. Phenotypic analysis showed that drug treatment enhanced the expression of negative co-stimulators PD-1, CTLA-4 and the transcription factor FoxP3 and decreased the expression of positive co-stimulators CD27 and CD28, whereas CD25 was unaffected. Mycophenolic acid-treated cells were anergic, but not suppressive, and at the same time proved hyperblastoid with high metabolic activity. Moreover, a reduced Akt/mTOR and STAT5 signaling was observed. Interestingly, the co-stimulatory molecule CD70 was uniquely and dose-dependently upregulated on mycophenolic acid-treated T cells and found to be directly linked to target enzyme inhibition. CD70 on mycophenolic acid-treated cells proved functional: an anti-CD70 agonist was found to restore both STAT5 and Akt/mTOR signaling and may thereby prevent apoptosis and promote survival. These novel insights may contribute to optimization of protocols for MPA-based immunosuppressive regimens.

Melanoma inhibitory activity, a biomarker related to chondrocyte anabolism, is reversibly suppressed by proinflammatory cytokines in rheumatoid arthritis.

OBJECTIVE: In mice, melanoma inhibitory activity (MIA) is a chondrocyte-specific molecule with similar regulation to collagen type II. As MIA is a small secreted protein, its value as cartilage biomarker in human inflammatory arthritis was assessed. METHODS: MIA tissue distribution was studied by quantitative PCR and immunohistochemistry. The regulation of MIA production was studied in vivo in rheumatoid arthritis (RA) (n = 37) and spondyloarthritis (SpA) (n = 30) synovial fluid (SF), and in vitro in alginate embedded human chondrocytes. Therapeutic modulation of serum MIA was evaluated during tumour necrosis factor (TNF)alpha and interleukin (IL)1 blockade in RA. RESULTS: MIA was primarily expressed by chondrocytes in the human joint. SF MIA levels were lower in RA than in SpA despite similar levels of overall synovial inflammation. Further analysis indicated that these levels were inversely correlated with the degree of joint inflammation in RA, but not in SpA, and that the levels of TNFalpha and IL1beta were significantly increased in RA versus SpA. Accordingly, these proinflammatory cytokines suppressed MIA mRNA and protein in cultured chondrocytes. This suppression was paralleled by suppression of cartilage anabolism as assessed by collagen type 2 and aggrecan mRNA. Treatment of patients with RA with TNF blockade or IL1 blockade induced an increase of serum MIA levels. CONCLUSION: The decreased levels of MIA in the inflamed RA joint and the coregulation of MIA and cartilage matrix molecules by proinflammatory cytokines indicate that joint inflammation in RA not only drives accelerated cartilage degradation but also suppresses cartilage anabolism. This inflammation-driven suppression is reversible in vivo.

A potent and selective p38 inhibitor protects against bone damage in murine collagen-induced arthritis: a comparison with neutralization of mouse TNFalpha.

BACKGROUND AND PURPOSE: The p38 kinase regulates the release of proinflammatory cytokines including tumour-necrosis factor-alpha (TNFalpha) and is regarded as a potential therapeutic target in rheumatoid arthritis (RA). Using the novel p38 inhibitor Org 48762-0, we investigated the therapeutic potential of p38 inhibition and compared this to anti-mouse (m)TNFalpha antibody treatment in murine collagen-induced arthritis (CIA). EXPERIMENTAL APPROACH: Pharmacological profiles of Org 48762-0 were characterized in kinase assays, cellular assays and in lipopolysaccharide (LPS)-induced inflammation in mice. The effects of Org 48762-0 and of mTNFalpha-neutralization on established arthritis were examined in murine CIA. KEY RESULTS: Org 48762-0 potently inhibited p38alpha kinase with a high degree of kinase selectivity. In cellular assays, Org 48762-0 reduced LPS-induced TNFalpha release. Oral administration of Org 48762-0 in mice showed drug-like pharmacokinetic properties and inhibited LPS-induced cytokine production. These pharmacological characteristics of Org 48762-0 prompted a comparison of therapeutic efficacy with mTNFalpha-neutralization in CIA. Org 48762-0 and anti-mTNFalpha antibody treatment equally inhibited development of arthritis when evaluated macroscopically. Radiological analyses revealed protection against bone damage for both treatments, although statistical difference was reached with Org 48762-0 treatment only. Further, micro-computed tomographical and histopathological analyses confirmed the protective effects of Org 48762-0 on joint damage. CONCLUSIONS AND IMPLICATIONS: Pharmacological targeting of p38 kinase provided good protection against joint tissue damage in CIA. In our experiments, neutralization of mTNFalpha produced less prominent suppression of bone damage. Our data suggest a therapeutic potential for selective and potent p38 inhibitors in RA.