Can baseline ultrasound results help to predict failure to achieve DAS28 remission after 1 year of tight control treatment in early RA patients?

BACKGROUND: At present, there are no prognostic parameters unequivocally predicting treatment failure in early rheumatoid arthritis (RA) patients. We investigated whether baseline ultrasonography (US) findings of joints, when added to baseline clinical, laboratory, and radiographical data, could improve prediction of failure to achieve Disease Activity Score assessing 28 joints (DAS28) remission (<2.6) at 1 year in newly diagnosed RA patients. METHODS: A multicentre cohort of newly diagnosed RA patients was followed prospectively for 1 year. US of the hands, wrists, and feet was performed at baseline. Clinical, laboratory, and radiographical parameters were recorded. Primary analysis was the prediction by logistic regression of the absence of DAS28 remission 12 months after diagnosis and start of therapy. RESULTS: Of 194 patients included, 174 were used for the analysis, with complete data available for 159. In a multivariate model with baseline DAS28 (odds ratio (OR) 1.6, 95% confidence interval (CI) 1.2-2.2), the presence of rheumatoid factor (OR 2.3, 95% CI 1.1-5.1), and type of monitoring strategy (OR 0.2, 95% CI 0.05-0.85), the addition of baseline US results for joints (OR 0.96, 95% CI 0.89-1.04) did not significantly improve the prediction of failure to achieve DAS28 remission (likelihood ratio test, 1.04; p = 0.31). CONCLUSION: In an early RA population, adding baseline ultrasonography of the hands, wrists, and feet to commonly available baseline characteristics did not improve prediction of failure to achieve DAS28 remission at 12 months. TRIAL REGISTRATION: Clinicaltrials.gov, NCT01752309 . Registered on 19 December 2012.

Induction therapy with a combination of DMARDs is better than methotrexate monotherapy: first results of the tREACH trial.

OBJECTIVE: To determine the most effective induction disease-modifying antirheumatic drug (DMARD) strategy in early rheumatoid arthritis (RA), second to compare one single dose of intramuscular glucocorticoids (GCs) with daily oral GCs during the induction phase. METHODS: The 3-month data of a single-blinded clinical trial in patients with recent-onset arthritis (tREACH) were used. Patients were included who had a high probability (>70%) of progressing to persistent arthritis, based on the prediction model of Visser. Patients were randomised into three induction therapy strategies: (A) combination therapy (methotrexate (MTX) + sulfasalazine + hydroxychloroquine) with GCs intramuscularly; (B) combination therapy with an oral GC tapering scheme and (C) MTX with oral GCs similar to B. A total of 281 patients were randomly assigned to strategy (A) (n=91), (B) (n=93) or (C) (n=97). RESULTS: The Disease Activity Score (DAS) after 3 months was lower in patients receiving initial combination therapy than in those receiving MTX monotherapy (0.39 (0.67 to 0.11, 95% CI)). DAS did not differ between the different GC bridging treatments. After 3 months 50% fewer biological agents were prescribed in the combination therapy groups. Although the proportion of patients with medication adjustments differed significantly between the treatment arms, no differences were seen in these adjustments due to adverse events after stratification for drug. CONCLUSION: Triple DMARD induction therapy is better than MTX monotherapy in early RA. Furthermore, no differences were seen in medication adjustments due to adverse events after stratification for drug. Intramuscular and oral GCs are equally effective as bridging treatments and both can be used.

Induction of phase I and II drug metabolism in rat small intestine and colon in vitro.

The aim of this study was to evaluate drug metabolism in rat small intestinal and colon precision-cut slices during 24 h of incubation and the applicability of these slices for enzyme induction studies. Various parameters were evaluated: intracellular levels of ATP (general viability marker), alkaline phosphatase activity (specific epithelial marker), villin expression (specific epithelial marker), and metabolic rates of 7-ethoxycoumarin (CYP1A), testosterone (CYP3A and CYP2B), and 7-hydroxycoumarin (glucuronide and sulfate conjugation) conversions. ATP and villin remained constant up to, respectively, 5 and 8 h in small intestine and up to 24 h in colon. The metabolic rate remained constant in small intestinal slices up to 8 h and decreased afterward to 24 to 92%, depending on the substrate studied. The inducibility of metabolism in small intestinal and colon slices was tested with several inducers at various concentrations and incubation times. The following inducers were used: 3-methylcholanthrene, beta-naphthoflavone, indirubin, and tert-butylhydroquinone (aryl hydrocarbon receptor ligands), dexamethasone (glucocorticoid receptor/pregnane X receptor ligand) and phenobarbital (constitutive androstane receptor ligand). After incubation with inducers, metabolic rates were evaluated with 7-ethoxycoumarin and testosterone (phase I) and 7-hydroxycoumarin (phase II) as substrate. All inducers elevated the metabolic rates consistent with the available published in vivo induction data. Induction of enzyme activity was already detectable after 5 h (small intestine) and after 8 h (colon) for 3-methylcholanthrene and beta-naphthoflavone and was clearly detectable for all tested inducers after 24 h (up to 20-fold compared with noninduced controls). In conclusion, small intestinal and colon precision-cut slices are useful for metabolism and enzyme induction studies.

Prediction of whole-body metabolic clearance of drugs through the combined use of slices from rat liver, lung, kidney, small intestine and colon.

1: The aim was to investigate whether precision-cut rat tissue slices could be used to predict metabolic drug clearance in vivo. To obtain a complete picture, slices not only from liver, but also from lung, kidney, small intestine and colon were included. 2: The metabolic clearances of 7-ethoxycoumarin, 7-hydroxycoumarin, testosterone, methyltestosterone and warfarin were determined by measuring the disappearance of these compounds during incubation with slices prepared from liver, lung, kidney, small intestine and colon. 3: The total in vitro metabolic clearance was determined by adding the individual in vitro organ clearances from the slices. Prediction based on the in vitro clearance was within an order of magnitude to the corresponding in vivo values. Interestingly, the relative contribution of extrahepatic metabolic clearance of the studied compounds to total clearance was remarkably high, ranging from 35 to 72% of the total metabolic clearance. 4: It is concluded that the model of multi-organ precision-cut slices is a useful in vitro tool for prediction of in vivo metabolic clearance. In addition, it provides information about the relative contribution of the liver, lung, kidney, small intestine and colon to the total metabolic clearance.

Drug-metabolizing activity of human and rat liver, lung, kidney and intestine slices.

1. Organ-specific biotransformation was studied in human and rat liver, lung, kidney and small intestine slices and compared on a protein basis, using four model substances. 2. Deethylation of lidocaine was highest in liver slices from both man and rat, followed by the small intestine. 3. Metabolism of testosterone was highest in liver slices, but a different overall metabolic pattern was found between the different organs. 4. Lung, kidney and intestine slices prepared from human and rat organs showed mainly an unknown metabolite of 7-ethoxycoumarin identified as 4-ethoxy-2-hydroxyphenyl propionic acid (EPPA). 5. The maximal metabolism of 7-ethoxycoumarin in slices was equal with in vivo V(max) in the rat. 6. Phase II metabolism of 7-hydroxycoumarin in kidney and intestinal slices was about 60% of the activity in liver slices. 7. In conclusion, organs other than the liver show a surprisingly high drug-metabolizing activity. Thus, the use of precision-cut slices of a combination of drug metabolizing organs in an in vitro test system from both animal and human origin is required for a proper systematic prediction of drug metabolism in man.

Rat liver slices as a tool to study LPS-induced inflammatory response in the liver.

BACKGROUND/AIMS: Inflammation in the liver is a complex interaction between parenchymal and non-parenchymal cells, and therefore can not be studied in vitro in pure cultures of these cells. METHODS: We investigated whether Kupffer cells in the liver slice are still responsive to an inflammatory stimulus of lipopolysaccharide (LPS), and evoke an inflammatory response in the hepatocytes. RESULTS: TNFalpha, IL-1beta and IL-10 were significantly elevated in culture medium of LPS-stimulated rat liver slices. Nitric oxide (NO) production of LPS-treated slices gradually increased from 5 to 24 h (24 h: 81+/-5 microM vs. 14+/-2 microM in control P < 0.05), paralleled by inducible nitric oxide synthase (iNOS) in the hepatocytes, iNOS mRNA was induced after 3 h. NO production but not iNOS induction was significantly inhibited by NOS inhibitors S-methylisothiourea and N(G)-nitro-L-arginine methylester. Both pentoxifylline and dexamethasone inhibited TNFalpha and IL-1beta production, albeit to a different extent, iNOS induction and, as a result thereof, NO production. CONCLUSIONS: These results imply that non-parenchymal cells in liver slices are viable and can be activated by LPS. In addition, it is concluded that the upregulation of iNOS in hepatocytes by LPS is caused by cytokines produced by Kupffer cells because inhibition of TNFalpha and IL-1beta production attenuated iNOS induction.

The applicability of rat and human liver slices to the study of mechanisms of hepatic drug uptake.

In the present study we investigated the applicability of the liver slice model to study mechanisms of drug uptake. Four model compounds were investigated that enter hepatocytes via entirely different membrane transport mechanisms. Rhodamine B (RB), which enters hepatocytes by passive diffusion, was homogeneously distributed throughout the rat liver slice (250 microm thickness) within 5 min, indicating that the penetration rate into the slice and the diffusion rate into the cells are rapid. In contrast, lucigenin (LU), which is taken up by hepatocytes through adsorptive endocytosis, was detected in the inner cell layers after 15 min. Digoxin uptake into the slice showed a temperature-dependent component and was stereoselectively inhibited by quinine, which is compatible with the involvement of a carrier-mediated uptake mechanism. The neo-glycoalbumin Lactose(27)-Human Serum Albumin (Lact(27)-HSA) and the negatively charged Succinylated-Human Serum Albumin (Suc-HSA) entered the slices and were taken up temperature-dependently into hepatocytes and endothelial cells, respectively. The liver slice preparation is a valuable tool to investigate the mechanisms of cellular uptake of drugs. Moreover, the precision-cut liver slices offer the unique possibility to study both hepatocyte and endothelial cell function in human and rat liver.

Organ slices as an in vitro test system for drug metabolism in human liver, lung and kidney.

Metabolism of xenobiotics occurs mainly in the liver, but in addition, the lungs and kidneys may contribute considerably. The choice of the animal species during drug development as a predictive model for the human condition is often inadequate due to large interspecies differences. Therefore, a universal method for the preparation and incubation of human and animal liver, lung and kidney tissue is being developed for drug metabolism and toxicity testing using precision-cut organ slices. Human tissue was obtained from surgical waste material. Slices were made from rat and human liver, kidney and agar-filled (1.5%, w/v) lung tissue using a Krumdieck tissue slicer and incubated in six-well plates. The morphology and the ATP content show that viability is maintained during 3 hours of incubation. These organ slices show a variety of phase I (hydroxylation, oxidation and O- and N-deethylation) and phase II (glucuronidation and sulfation) metabolic routes using lidocaine, testosterone, 7-ethoxycoumarin and 7-hydroxycoumarin as substrates. The metabolic patterns and rates were found to be different for the various organs and species studied. The use of human tissue slices will enable us to collect more human-specific data on drug metabolism and toxicity. This may lead to a more adequate choice of animal species used during drug development and will result in a considerable reduction in the use of experimental animals.

Effect of cold and warm ischaemia on drug metabolism in isolated hepatocytes and slices from human and monkey liver.

1. The influence of short-term cold storage in University of Wisconsin organ preservation solution (UW) on the ability to metabolize lidocaine, testosterone and 7-ethoxycoumarin in isolated human and cynomolgus monkey (Macaca fascicularis) hepatocytes and liver slices has been investigated. 2. The human liver tissue was obtained from two different sources, i.e. healthy liver tissue from patients undergoing partial hepatectomy because of metastases of colorectal carcinoma (PH livers) and donor tissue remaining as surgical waste after reduced size or split liver transplantation (Tx livers). Tx livers were perfused in situ with ice-cold UW avoiding warm ischaemia. This in contrast with PH livers, where the operation caused warm ischaemia for 5-90 min. 3. Liver slices and hepatocytes from cynomolgus monkey liver showed comparable metabolic rates for the substrates tested, indicating that all hepatocytes in the slice are participating in the biotransformation of the substrates. These monkey liver preparations can be stored up to 18 h with only a slight loss of their metabolic capacity. 4. Liver slices and isolated hepatocytes from the Tx livers as well as isolated cells from the PH livers could also be stored up to 18 h without losing metabolic capacity. However, for liver slices prepared from PH livers cold storage is not recommended, because metabolic function was reduced by approximately 40% after 18 h.

Inhibitory effect of cyclosporin A on antigen and alloantigen presenting capacity of human epidermal Langerhans cells.

The effect of cyclosporin A (CyA) on the capacity of human epidermal Langerhans cells (LC) to stimulate allogeneic T cells or to present antigen to autologous T cells was investigated. Preparations of LC enriched by discontinuous density gradient centrifugation were pulsed for 2 or 16 h with graded doses (5-5000 ng/ml) of CyA prior to co-culture with T cells. Pretreatment of LC with CyA resulted in a dose-dependent decrease of the functional capacity of LC to stimulate T cells. This inhibition (up to 90%), already achieved after a pulse of 2 h, was not due to a cytotoxic effect of the drug and appeared to be reversible. The possibility that CyA exerted its effect indirectly on T cells via release of CyA from LC into the supernatant during co-culture was excluded. The suppression of immunostimulatory function was a direct effect of the drug on LC. CyA did not affect the production by LC of IL-1 or prostaglandin, nor the expression of MHC class II products HLA-D and RFD1 or adhesion molecules ICAM-1 and LFA-3. These results suggest that inhibition of contact allergic skin reactions by CyA may be due in part to an impairment of the function of LC.

Antibody response to human immunodeficiency virus type 1 protease according to risk group and disease stage.

Three groups with different routes of human immunodeficiency virus type 1 (HIV-1) transmission (homosexual men, hemophiliacs, and children) were studied for serum antibodies to a recombinant form of the HIV-1 protease using an enzyme-linked immunoassay. At 1 year after seroconversion, defined as the moment antibodies to HIV-1 proteins were first detected, 56% (34/61) of the homosexual men had antibodies to protease, and 2 years after seroconversion this percentage was 63% (24/38). Within this 2-year period these antibodies were no longer detected in 16% (9/56). A similar pattern was observed in 20 hemophiliacs who seroconverted after exposure to HIV-1-contaminated blood products. We found that 63% (160/255) of homosexual men in Centers for Disease Control stage II or III, 60% (9/15) of patients with acquired immunodeficiency syndrome (AIDS)-related complex, and 36% (14/39) of patients with AIDS had antibodies to protease. In 255 homosexual men in Centers for Disease Control stage II or III, antibodies to protease were significantly more frequently found in samples lacking HIV-1 antigen (P less than 0.001) and possessing antibodies to HIV-1 core proteins (P less than 0.001). Twenty-four persons who developed AIDS were studied longitudinally: 58% (14/24) had antibodies to protease 1 year before developing symptoms; 29% (7/24) showed a decline and 29% (7/24) showed a loss of antibodies to protease at the onset of symptoms. Within a group of 47 HIV-1-infected children, 90% (18/20) with a stable disease course were persistently protease antibody positive, versus 4 of 27 children (15%) with an unstable disease course (P = 0.0001). These data indicate that HIV-1 protease is expressed and antigenic in most HIV-1-infected individuals and that a decline or absence of antibodies to protease is strongly associated with unstable disease in children and AIDS in adults.