Signal transduction and metabolism in chondrocytes is modulated by lactoferrin.

OBJECTIVE: Activation of granulocytes causes a considerable rise in the concentration of lactoferrin (Lf) in synovial fluid (SF). We here investigate consequences thereof on signal transduction and the balance between catabolic and anabolic metabolism in chondrocytes. METHODS: Signal transduction was analysed in cultured chondrocytes by immunodetection of mitogen activated protein kinases (MAPK) and analysis of Smad2 translocation to the nucleus. Expression levels of matrix metalloproteinases (MMPs) and of aggrecan were measured by reverse-transcription-PCR. The proteolytic activity of MMPs was ascertained by zymography. Expression of the low-density-lipoprotein-receptor-related-protein-1 (LRP-1), a Lf receptor for signalling, was assayed by immunohistochemistry in cartilage and in cultured chondrocytes by immunoblotting. RESULTS: We found LRP-1 expressed in dedifferentiated chondrocytes in culture and in cartilage tissue preferentially on the articular surface where it can encounter Lf within SF. Lf stimulated proliferation of chondrocytes, comparable to transforming growth factor-beta1 (TGFbeta1) and activated p38 and the extracellular-signal regulated-kinases 1/2 (ERK1/2) within minutes. Surprisingly, Lf induced nuclear Smad2 translocation, a signal pathway ascribed to TGFbeta receptor activation. Lf significantly increased the levels of catabolic indicators such as MMP1, MMP2, MMP3 and MMP13 and inhibited aggrecan synthesis. CONCLUSION: Lf is a robust regulator of chondrocyte metabolism, comparable to TGFbeta1. The catabolic influence together with the proliferative stimulus indicates a function as an early phase cytokine, enhancing MMPs, necessary for degradation of damaged tissue and stimulating proliferation of chondrocytes, necessary for reconstruction.

Drug monitoring and viral response to lopinavir/ritonavir or saquinavir/ritonavir containing regimens in individuals infected with the human immunodeficiency virus type 1.

The aim of this study was to correlate results of therapeutic drug monitoring, genotypic resistance and viral response to lopinavir/ritonavir (LPV/r) or saquinavir/ritonavir (SQV/r) containing antiretroviral regimens. The retrospective short-term study included 20 patients with LPV/r and 20 patients with SQV/r containing highly active antiretroviral therapy (HAART). At baseline 7 LPV/r patients and 10 SQV/r patients had CD4+T cell counts above 410 cells/microl. After 6 months CD4+T cells had doubled in 5 LPV/r and 2 SQV/r patients. In LPV/r patients the mean serum concentration of lopinavir (LPV) was 2.6 ppm and 67% of all LPV/r samples had 50 or fewer viral copies/ml. In SQV/r patients the mean serum concentration of saquinavir (SQV) was 2.1 ppm. 79% of all SQV/r samples had 50 or fewer viruses/ml. Pharmacoenhanced regimens efficiently suppress human immunodeficiency virus type 1 (HIV-1) and the risk of developing resistance mutations is therefore reduced. The implementation of drug monitoring is an additional tool to determine optimal treatment conditions.

Capillary electrophoretic separation of protease inhibitors used in human immunodeficiency virus therapy.

The scope of this work was to investigate the migration behavior of the currently used protease inhibitors for antiretroviral therapy of people infected with the human immunodeficiency virus and to develop a method for their capillary electrophoretic separation and determination. All of the protease inhibitors (indinavir, saquinavir, nelfinavir, amprenavir, and ritonavir) contain at least one basic amino functional group. As a consequence, they can be separated by capillary zone electrophoresis using acidic buffer electrolytes. A fast electroosmotic flow is established in order to increase separation speed, by adding a cationic electroosmotic flow modifier to the electrolyte. After using conventional serum pretreatment procedures it is possible to separate all five protease inhibitors within less than 5 min. In addition, a non-aqueous CE method is also presented which enables the separation of three protease inhibitor compounds within less than 3 min.

Assorted effects of TGFbeta and chondroitinsulfate on p38 and ERK1/2 activation levels in human articular chondrocytes stimulated with LPS.

OBJECTIVES: Inadequate cellular response of chondrocytes to stress frequently terminates in osteoarthritis (OA). Adequate response is fundamentally modulated by concerted cytokine signaling events, directing degradation and synthesis of cartilage on articular surfaces where and whenever necessary. Transforming growth factor (TGF)beta is a prominent mediator in cartilage anabolism, although particular catabolic activities are occasionally reported. Clearly, before the TGFbeta signal gets through to the gene regulatory machinery, cross talk with modulators occurs. METHOD: We tested the hypothesis whether chondroitinsulfate (CS) modulates cell signaling. TGFbeta and/or soluble CS was added to human articular chondrocytes (HACs) and activation of p38 and extracellular signal related kinase (ERK)1/2 was determined by immunoblot analysis. Expression levels of mRNA of matrix metalloproteinase (MMP)-2, -3 and -13 were determined by real-time polymerase chain reaction (PCR). RESULTS: No significant effects were observed unless cells were stimulated with lipopolysaccharide (LPS), invigorating catabolic metabolism in chondrocytes. LPS effects, however, were profoundly modulated by TGFbeta, CS and both applied in combination. Most prominent, the silencing of p38 stress signal by CS was superimposable to that of TGFbeta. Phospho-ERK1/2 levels were raised by TGFbeta three-fold over LPS induced levels. In contrast, CS treatment, alone or combined with TGFbeta, reduced phosphorylation significantly below LPS induced levels. Finally, suppression of LPS induced MMP-13 mRNA levels resulted with CS. CONCLUSION: Soluble CS modulates signaling events in chondrocytes concurrent with MMP-13 down regulation. The effects observed suggest a feedback signaling mechanism cross talking with TGFbeta-signal pathways and may serve an explanation, on the cellular level, for the beneficial effects found in clinical studies with pharmacologic application of CS.

Fast separation of underivatized carbohydrates by coelectroosmotic capillary electrophoresis.

A method for the rapid quantitative analysis of underivatized acidic sugars, monosaccharides and disaccharides using coelectroosmotic capillary electrophoresis was developed. Indirect UV detection at 254 nm using sorbate as background electrolyte was employed for monitoring the analytes. A highly alkaline pH value of the electrolyte system was chosen in order to achieve an electrophoretic mobility of the saccharides towards the anode. A dynamic reversal of the electroosmotic flow and, by this means, a codirectional movement of the negatively charged analytes and the electroosmotic flow is accomplished by employing a polycationic surfactant (hexadimethrine bromide), which is added to the background electrolyte. To further improve the resolution of specific carbohydrates, acetone is used as organic modifier. A practical application of the developed method for the fast determination of fructose, glucose, and sucrose in various soft drinks is provided.

Separation of cresols using coelectroosmotic capillary electrophoresis.

A new method of capillary electrophoresis was established to separate the three cresol isomers. Cresols are of importance due to their use as insecticides, precursors of various dye intermediates, resins, fire-resistant fluids and other purposes. Reversion of the electroosmotic flow was carried out by a polycationic ammonium compound. The separations were carried out at high pH values (pH 12) to obtain complete dissociation of the cresols and short times of analysis (less than 5 min) through fast migration of the cresol towards the anode. Various aliphatic alcohols were added to improve peak shape and resolution.

Anion-exchange capillary electrochromatography with indirect UV and direct contactless conductivity detection.

Conductivity detection is applied to ion-exchange capillary electrochromatography (IE-CEC) with a packed stationary phase, using a capacitively coupled contactless conductivity detector with detection occurring through the packed bed. Columns were packed with a polymeric latex-agglomerate anion-exchanger (Dionex AS9-SC). A systematic approach was used to determine suitable eluants for IE-CEC separations using simultaneous indirect UV and direct conductivity detection. Salicylate and p-toluenesulfonate were identified as potential eluant competing anions having sufficient eluotropic strength to induce changes in separation selectivity, but salicylate was found to be unsuitable with regard to baseline stability. It was also found for both indirect UV and direct conductivity detection that homogenous column packing was imperative, and monitoring of the baseline could be used to assess the homogeneity of the packed bed. Using a p-toluenesulfonate eluant, the separation of eight common anions was achieved in 2.5 min. Direct conductivity detection was found to be superior to indirect UV detection with regard to both baseline stability and detection sensitivity with detection limits of 4-25 microg/L being obtained. However, the calibration for each anion was not linear over more than one order of magnitude. When using conductivity detection, the concentration of the eluant could be varied over a wider range (2.5-50 mM p-toluenesulfonate) than was the case with indirect UV detection (2.5-10 mM), thereby allowing greater changes in separation selectivity to be achieved. By varying the concentration of p-toluenesulfonate in the eluant, the separation selectivity could be manipulated from being predominantly ion-exchange in nature (2.5 mM) to predominantly electrophoretic in nature (50 mM).

Capillary electrophoresis and contactless conductivity detection of ions in narrow inner diameter capillaries.

Capillary electrophoresis and conductometry represent a combination of a high-resolution separation method with a sensitive detection principle for the analysis of ionic species. In this paper, results are reported that are obtained with a contactless conductivity detector. This device works without a galvanic contact of the electrolyte and the electrodes. The conductivity sensor is based on two metal tubes that act as cylindrical capacitors. These electrodes are both placed around a fused-silica capillary with a detection gap of 1 mm left in between. When a high audio or low ultrasonic oscillation frequency between 40 and 100 kHz is applied to one of the electrodes, a signal is produced as soon as an analyte zone with a different conductivity compared to the background electrolyte passes the detection gap. An amplifier and rectifier is connected to the other electrode where the signal is further processed. Limits of detection for lithium and fluoride are 4 and 13 ppb, respectively, with a linear range over 4 orders of magnitude from 90 ppb up to more than 1000 ppm for both anions and cations. Furthermore, it is demonstrated that for species with lower equivalent conductivities, such as organic ions, indirect conductivity detection is a sensitive alternative to indirect optical detection methods. Limits of detection of 50 ppb and below are obtained for organic acids.

Contactless conductivity detection for capillary electrophoresis.

A contactless capacitively coupled conductivity detector for capillary electrophoresis is introduced. The detector consists of two electrodes which are placed cylindrically around the outer polyimide coating of the fused-silica capillary with a detection gap of 2 mm. The electrodes form a cylindrical capacitor, and the electric conductivity of the solution in the gap between the electrodes is measured. A high audio or low ultrasonic frequency for coupling of the ac voltage is used in order to minimize the influence of reactance of the liquid. For an improved version of the detector, two syringe cannulas are used as the electrodes and the capillary is simply assembled into the tubing. This allows an easy placement of the detector on various positions along the capillary. The limit of detection of inorganic cations and anions is 200 ppb, as determined for sodium and chloride, respectively.