MIF attenuates the suppressive effect of dexamethasone on IL-6 production by nasal polyp.

BACKGROUND: Nasal polyposis (NP) is a chronic inflammatory disease of the upper airways, that characterized by inflammatory cells infiltration, extracellular matrix accumulation and oedema. Interleukin-6 (IL-6) is a multifunctional cytokine, implicated in various inflammatory conditions, including NP pathogenesis. Macrophage migration inhibitory factor (MIF) is a pro-inflammatory mediator able to antagonize the inhibitory effects of glucocorticoids on the expression of various cytokines and growth factors. AIM: To investigate the presence of MIF in nasal polyp tissues and the influence of a MIF activity inhibitor on dexamethasone effects on IL-6 production. PATIENTS AND METHODS: Nasal polyps were resected by functional endoscopic sinus surgery for treatment of chronic sinusitis with polyposis and healthy nasal mucosa was taken during nasal septoplasty-chochoplasty. MIF and IL-6 levels were determined by ELISA. The expression of MIF and IL-6 at the mRNA level was ascertained by RT-PCR. RESULTS: MIF was detected in all polyp tissue extracts and tissue cultures conditioned media. MIF and IL-6 expression were significantly higher in polyp tissues as compared to normal nasal mucosa tissues. Dexamethasone at concentration 1-100 microM caused a statistically significant dose-dependent suppression of IL-6 production by polyp tissue cultures. Inhibition of MIF by (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), an inhibitor of MIF tautomerase activity, significantly enhanced the dexamethasone suppressive effect on IL-6 production. CONCLUSIONS: MIF, presence in polyp tissue, attenuates the suppressive effect of dexamethasone on the production of IL-6 by this tissue, since the simultaneous use of its inhibitor ISO-1 leads to an enhancement of dexamethasone activity. Therefore, it is reasonable to propose that the utilization of MIF inhibitors together with glucocorticoids in clinical practice may be beneficial in the treatment of NP.

Presence of hyaluronidase isoforms in nasal polyps.

BACKGROUND: Nasal polyps are benign lesions originating from the nasal mucosa or paranasal sinuses. The most important etiological factor seems to be increased hydration of epithelium and hyperplasia of the extracellular matrix, which may involve hyaluronan, a high molecular mass extracellular glycosaminoglycan. Degradation of hyaluronan proceeds through the action of specific hyaluronidases. OBJECTIVE: The aim of the present study was to investigate the hydrodynamic size of hyaluronan and the presence of the various hyaluronidase isoforms in nasal polyps. METHODS: Samples of polypoid mucosal tissue and normal nasal mucosa were obtained from twenty patients suffering from nasal polyposis. Zymographic analysis and western blotting were used to detect hyaluronidase activity. RESULTS: The results indicated the presence of hyaluronan of small molecular mass in all samples examined. About one third of it has a mean molecular mass of 240 kDa, exactly that required for the expression of inflammatory response. Laboratory analysis suggested that degradation of hyaluronan occurred through the action of three hyaluronidase isoforms: Hyal-1, Hyal-2 and PH-20. CONCLUSIONS: Since hyaluronan fragments of 200-250 kDa induce the expression of inflammatory cytokines, a specific role of hyaluronidases in the development or progression of nasal polyps may be concluded. Therefore, new treatment protocols may be proposed.

Targeting the tumor proteasome as a mechanism to control the synthesis and bioactivity of matrix macromolecules.

Extracellular matrices (ECMs) are dynamic structures that provide cells not only with a structural support but, importantly, exhibit significant functional roles in the control of key cellular events such as adhesion, migration, proliferation, differentiation, and survival. In tumors, matrix effectors such as proteoglycans (PGs) and matrix metalloproteinases (MMPs) constitute major regulators of the interactions between tumor cells and their microenvironment and, therefore, they have been identified as potential molecular targets that are expected to advance the pharmacological treatment of cancer. ECMs composition is highly affected by cells through intrinsic regulatory mechanisms, such as the ubiquitin-proteasome system (UPS). Proteasome is a major cellular protease complex that controls the concentration and turnover of molecules in ECMs, including certain types of PGs, MMPs and collagens, and consequently, in the tumor microenvironment. Furthermore, proteasome activity is regulated by PG-derived intracellular glycosaminoglycan moieties revealing a critical inter-dependence of these compounds. Since ECMs renewal and degradation can be tightly regulated by proteasome activities, its modulation may be considered as a novel strategy to control the properties of tumor microenvironment. Currently, there are several proteasome inhibitors targeting distinct molecular pathways either approved or in clinical trials for the treatment of multiple cancers. In this review, the novel approach of targeting the proteasome to selectively regulate the synthesis and the bioactivity of certain matrix PGs and MMPs is presented and discussed.

Dynamics of proteins: light scattering study of dilute and dense colloidal suspensions of eye lens homogenates.

We report a dynamic light scattering study on protein suspensions of bovine lens homogenates at conditions (pH and ionic strength) similar to the physiological ones. Light scattering data were collected at two temperatures, 20 and 37 degrees C, over a wide range of concentrations from the very dilute limit up to the dense regime approaching the physiological lens concentration. A comparison with experimental data from intact bovine lenses was advanced, revealing differences between dispersions and lenses at similar concentrations. In the dilute regime, two scattering entities were detected and identified with the long-time self-diffusion modes of alpha-crystallins and their aggregates, which naturally exist in lens nucleus. Upon increasing protein concentration, significant changes in time correlation function were observed starting at approximately 75 mg ml(-1), where a new mode originating from collective diffusive motions becomes visible. Self-diffusion coefficients are temperature insensitive, whereas the collective diffusion coefficient depends strongly on temperature revealing a reduction of the net repulsive interparticle forces with decreasing temperature. While there are no rigorous theoretical approaches on particle diffusion properties for multicomponent, nonideal hard sphere polydispersed systems, as the suspensions studied here, a discussion of the volume fraction dependence of the long-time self-diffusion coefficient in the context of existing theoretical approaches was undertaken. This study is purported to provide some insight into the complex light scattering pattern of intact lenses and the interactions between the constituent proteins that are responsible for lens transparency. This would lead to understand basic mechanisms of specific protein interactions that lead to lens opacification (cataract) under pathological conditions.

In vitro effects of non-steroidal anti-inflammatory drugs on cytokine, prostanoid and matrix metalloproteinase production by interface membranes from loose hip or knee endoprostheses.

OBJECTIVE: To study the effects of the non-steroidal anti-inflammatory drugs (NSAIDs) aceclofenac, piroxicam, tenoxicam and indomethacin on cytokine, matrix metalloproteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs) and prostaglandin E2 (PGE2) production, by interface membranes (IFT), obtained at revision surgery for aseptic loosening of total joint arthroplasty. Involvement of these soluble factors is well documented and probably, a pharmaceutically induced inhibition of them might retard loosening. METHODS: IFTs from 10 patients with a loose hip or knee endoprosthesis were collected. The possibility of septic loosening was thoroughly excluded by histopathologic and microbiologic evaluation. IFTs were cultured in the absence or presence of the tested drugs and the levels of the soluble mediators were determined, using electrophoretic and enzyme-linked immunosorbent assay techniques. Paracetamol was used as neutral drug. RESULTS: All NSAIDs exhibited a pronounced inhibitory effect upon the production of interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-alpha). This specific effect on IL-6 is reported in the literature for the first time. The majority of NSAIDs also induced the production of IL-1beta in an adequate portion of samples. These drugs did not have a clear effect on MMP synthesis, but they had a stimulatory tendency on TIMP-1 production. Paracetamol, significantly decreased the synthesis of TNF-alpha and that of the gelatinases. CONCLUSION: Our in vitro results are encouraging, since it appears that the action of NSAIDs, globally considered, may be beneficial upon the loosening process. The inhibitory effect of paracetamol upon TNF-alpha and gelatinases is intriguing. Our data, if supported by similar observations, probably justify performance of long-term clinical trials.

Diagnostic and classification value of metalloproteinases in squamous human laryngeal carcinoma.

Metalloproteinases (MMPs) are a class of enzymes largely involved in tumour progression and metastasis. At least twenty different enzymes are recognized that are also present under normal state of tissues. Their activity is regulated by their presence as proenzymes and by the concomitant presence of the respective tissue inhibitors (TIMPs). The present study describes the alterations of MMPs observed in human laryngeal carcinoma with respect to tumour classification and compares their activity in normal and cancerous tissues and biopsy specimens. Samples from five patients who underwent laryngectomy, from five biopsies and three from autopsies were used. The MMPs of normal and malignant human laryngeal cartilage and of biopsy specimens were identified immunochemically and by zymography using gelatin or casein as substrates. Healthy cartilage from autopsies was found to contain almost exclusively MMP-1, proMMP-2 and proMMP-9. Normal parts from laryngectomies contained, in addition, significant amounts of active MMP-2. The respective malignant parts contained both MMP-2 and -9 in increased amounts in their latent and active forms. Similar profile of MMPs was also identified in tissues surrounding affected cartilage. These alterations were found to be in good accordance with tumour stage and were also observed in biopsy samples. Thus, analysis of MMPs in biopsies can be used together with the clinicopathological parameters for the classification or early diagnosis of laryngeal tumours.

The overall pattern of cardiac contraction depends on a spatial gradient of myosin regulatory light chain phosphorylation.

Evolution of the human heart has incorporated a variety of successful strategies for motion used throughout the animal kingdom. One such strategy is to add the efficiency of torsion to compression so that blood is wrung, as well as pumped, out of the heart. Models of cardiac torsion have assumed uniform contractile properties of muscle fibers throughout the heart. Here, we show how a spatial gradient of myosin light chain phosphorylation across the heart facilitates torsion by inversely altering tension production and the stretch activation response. To demonstrate the importance of cardiac light chain phosphorylation, we cloned a myosin light chain kinase from a human heart and have identified a gain-in-function mutation in two individuals with cardiac hypertrophy.

Mixed echo train acquisition displacement encoding with stimulated echoes: an optimized DENSE method for in vivo functional imaging of the human heart.

Mixed echo train acquisition displacement encoding with stimulated echoes (meta-DENSE) is a phase-based displacement mapping technique suitable for imaging myocardial function. This method has been optimized for use with patients who have a history of myocardial infarction. The total scan time is 12-14 heartbeats for an in-plane resolution of 2.8 x 2.8 mm2. Myocardial strain is mapped at this resolution with an accuracy of 2% strain in vivo. Compared to standard stimulated echo (STE) methods, both data acquisition speed and resolution are improved with inversion-recovery FID suppression and the meta-DENSE readout scheme. Data processing requires minimal user intervention and provides a rapid quantitative feedback on the MRI scanner for evaluating cardiac function. Published 2001 Wiley-Liss, Inc.

Gelatinolytic and collagenolytic activity in periprosthetic tissues from loose hip endoprostheses.

OBJECTIVE: To study the contribution of different members of the metalloproteinases (MMP) family in gelatinolytic and collagenolytic potential, namely dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (DNP-S) sensitive proteolytic activity, in loose total hip arthroplasty (THA) endoprostheses. METHODS: Periprosthetic tissues and fluid samples were collected from patients subjected to hip endoprosthesis replacement. DNP-S sensitive proteolytic activity was evaluated by the degradation of synthetic DNP-S and reverse phase high performance liquid chromatography, while gelatinolytic activity was assessed by gelatin zymography. The isolation and separation of gelatinases was performed by gelatin- and concanavalin A-Sepharose affinity chromatographies and the identification of collagenases by immunoblot analysis. RESULTS: High gelatinolytic activity was observed in all periprosthetic tissue extracts and fluid samples. All samples also exhibited DNP-S degrading activity, without pretreatment by activating agents. Upon fractionation of MMP by gelatin-Sepharose affinity chromatography it was found that the gelatin-unbound collagenases are exclusively responsible for DNP-S degrading activity. Activated species of both MMP-1 and 13 were detected in most samples, but not the soluble form of MT1-MMP. Separation of gelatinases from each other and treatment with 4-aminophenylmercuric acetate (APMA) revealed that both enzymes mainly existed in complex with tissue inhibitor of metalloproteinase (TIMP). CONCLUSION: MMP-1 and MMP-13, which exist in activated form, could be responsible for the DNP-S-degrading activity in periprosthetic tissues and fluids, while the gelatinases do not contribute in this potential, since they mainly exist in complex with TIMP. The 2 collagenases may play a key role in the loosening of THA endoprostheses.

Imaging of urea using chemical exchange-dependent saturation transfer at 1.5T.

The purpose of this study was to screen for slow proton chemical exchange between water and kidney metabolites using a standard clinical 1.5-T scanner. Imaging was performed using a fast spin-echo sequence with a magnetization transfer (MT) preparation pulse train. Off-resonance saturation ranging from +/-50 to +/-1000 Hz was used on urea and urine phantoms and normal human subjects imaged through the kidneys. The positive frequency was used as the control for each frequency pair. Results of frequency sweeps show an asymmetric MT effect peaking at approximately 100 Hz ( thick similar1 ppm) for urea, urine, and renal parenchyma. Varying differences (5%-25%) occurred with different human subjects. Few differences were observed from phantom water or subject muscle tissue. Chemical exchange is detectable in the kidney near 1 ppm at 1.5 T, attributable to urea. This technique was used to produce in vivo distribution maps of this metabolite in vivo.

MRI of the human eye using magnetization transfer contrast enhancement.

PURPOSE: To determine the feasibility of using magnetization transfer contrast-enhanced magnetic resonance imaging (MRI) to track cataractous lens changes. METHODS: A fast spin-echo sequence was modified to include a magnetization transfer contrast (MTC) preparation pulse train. This consisted of twenty 8.5-msec sinc pulses, 1200 Hz upfield from the water resonance and 1.2-Hz power. The MTC preparation pulse was followed by acquisition through fast spin-echo imaging. The imaging parameters were number of excitations (NEX) = 1, echo time (TE) = 14 msec, recovery time (TR) = 2 sec, echo train length of eight echos, and a matrix size of 256 x 160. To reduce motion artifacts, the volunteers were asked to fixate on a blinking LED. Normal and MTC-enhanced images were acquired from normal volunteers and volunteers with nuclear or cortical cataracts. RESULTS: The eye was adequately imaged, with few motion artifacts appearing. The lens was well resolved, despite the short T(2). The cornea and ciliary body were also clearly visible. In the lens, resolution of the epithelium and cortex were enhanced with MTC. In addition, contrast-to-noise ratios were measured for each image. Examination of the contrast-to-noise ratio confirmed that MTC increased the contrast between the nucleus and cortex. Unenhanced MRIs showed significant differences between the cortex of normal volunteers and volunteers with cataracts. MTC-enhanced images improved the sensitivity to changes in the nucleus. CONCLUSIONS: In this preliminary study, we were able to use MTC-enhanced MRI to obtain high-contrast images of the human lens. Regular and enhanced MRIs detected statistically significant differences between normal and cataractous lenses.

In vivo (1)H double quantum filtered MRI of the human wrist and ankle.

Proton double quantum filtered (DQF) NMR imaging was applied in vivo to the human wrist and ankle with a clinical 1.5 T MR scanner. Water molecules having anisotropic motion were detected from tendons and ligaments. Images of Achilles tendon were obtained for a voxel size of 1.25 x 1.25 x 20 mm with three values of TR = 1.0, 0.5, and 0.2 sec, resulting in total acquisitions time of 17, 8.5, and 3.4 mins, respectively. Some degradation of the signal-to-noise ratio was observed at the shortest TR value and the contrast was significantly reduced due to SQ coherence leakage. The in vivo DQF images showed structure within the tendon that is otherwise not visible by conventional gradient-recalled echo (GRE) methods. Published 2000 Wiley-Liss, Inc.

A new class of contrast agents for MRI based on proton chemical exchange dependent saturation transfer (CEST).

It has been previously shown that intrinsic metabolites can be imaged based on their water proton exchange rates using saturation transfer techniques. The goal of this study was to identify an appropriate chemical exchange site that could be developed for use as an exogenous chemical exchange dependent saturation transfer (CEST) contrast agent under physiological conditions. These agents would function by reducing the water proton signal through a chemical exchange site on the agent via saturation transfer. The ideal chemical exchange site would have a large chemical shift from water. This permits a high exchange rate without approaching the fast exchange limit at physiological pH (6.5-7.6) and temperature (37 degrees C), as well as minimizing problems associated with magnetic field susceptibility. Numerous candidate chemicals (amino acids, sugars, nucleotides, heterocyclic ring chemicals) were evaluated in this preliminary study. Of these, barbituric acid and 5, 6-dihydrouracil were more fully characterized with regard to pH, temperature, and concentration CEST effects. The best chemical exchange site found was the 5.33-ppm indole ring -NH site of 5-hydroxytryptophan. These data demonstrate that a CEST-based exogenous contrast agent for MRI is feasible.

Effect of muscle action and metabolic strain on oxidative metabolic responses in human skeletal muscle.

A recent report suggests that differences in aerobic capacity exist between concentric and eccentric muscle action in human muscle (T. W. Ryschon, M. D. Fowler, R. E. Wysong, A. R. Anthony, and R. S. Balaban. J. Appl. Physiol. 83: 867-874, 1997). This study compared oxidative response, in the form of phosphocreatine (PCr) resynthesis rates, with matched levels of metabolic strain (i.e., changes in ADP concentration or the free energy of ATP hydrolysis) in tibialis anterior muscle exercised with either muscle action in vivo (n = 7 subjects). Exercise was controlled and metabolic strain measured by a dynamometer and (31)P-magnetic resonance spectroscopy, respectively. Metabolic strain was varied to bring cytosolic ADP concentration up to 55 microM or decrease the free energy of ATP hydrolysis to -55 kJ/mol with no change in cytoplasmic pH. PCr resynthesis rates after exercise ranged from 31.9 to 462.5 and from 21.4 to 405.4 micromol PCr/s for concentric and eccentric action, respectively. PCr resynthesis rates as a function of metabolic strain were not significantly different between muscle actions (P > 0.40), suggesting that oxidative capacity is dependent on metabolic strain, not muscle action. Pooled data were found to more closely conform to previous biochemical measurements when a term for increasing oxidative capacity with metabolic strain was added to models of respiratory control.

High-resolution strain analysis of the human heart with fast-DENSE.

Single breath-hold displacement data from the human heart were acquired with fast-DENSE (fast displacement encoding with stimulated echoes) during systolic contraction at 2.5 x 2.5 mm in-plane resolution. Encoding strengths of 0.86-1.60 mm/pi were utilized in order to extend the dynamic range of the phase measurements and minimize effects of physiologic and instrument noise. The noise level in strain measurements for both contraction and dilation corresponded to a strain value of 2.8%. In the human heart, strain analysis has sufficient resolution to reveal transmural variation across the left ventricular wall. Data processing required minimal user intervention and provided a rapid quantitative feedback. The intrinsic temporal integration of fast-DENSE achieves high accuracy at the expense of temporal resolution.

Analysis of N-acetyl and N-glycolylneuraminic acid in rat serum and tissues with Walker 256 carcinoma by high-performance liquid chromatography.

Serum and tissue specimens from healthy Wistar rats and from rats with Walker 256 carcinoma were analysed for N-acetyl and N-glycolylneuraminic acid by high performance liquid chromatography (HPLC) as per-O-benzoylated derivatives. Both neuraminic acids were identified, while N-acetylneuraminic acid was the predominant sialic acid. Samples from rats with generalized metastasis showed a significant increase (45-80%) of total sialic acids. This phenomenon in serum is caused by the overproduction of sialic acids, as a result of synthesis of both types of neuraminic acids to a similar molar ratio. The increase of sialic acids in rat bones with metastatic cancer is mainly because of increased N-acetylneuraminic acid synthesis. These results suggest that the molecular mechanisms responsible for cancer metastasis in different tissues may be closely associated with increased synthesis of dominating neuraminic acid.

DENSE: displacement encoding with stimulated echoes in cardiac functional MRI.

Displacement encoding with stimulated echoes (DENSE) was developed for high-resolution myocardial displacement mapping. Pixel phase is modulated by myocardial displacement and data spatial resolution is limited only by pixel size. 2D displacement vector maps were generated for the systolic action in canines with 0.94 x 1.9 mm nominal in-plane resolution and 2.3 mm/pi displacement encoding. A radial strain of 0.208 was measured across the free left ventricular wall over 105 ms during systole. DENSE displacement maps require small first-order gradient moments for encoding. DENSE magnitude images exhibit black-blood contrast which allows for better myocardial definition and reduced motion-related artifacts.

A solid-phase assay for quantitative analysis of sulfated glycosaminoglycans at the nanogram level. Application to tissue samples.

A sensitive and accurate solid-phase methodology for the quantitative analysis of glycosaminoglycans is described. Chondroitin-4-sulfate (CSA) was labelled with biotin hydrazide after the reaction of its carboxyl groups with it in the presence of carbodiimide. Polystyrene plates modified with sequential reaction with glutaraldehyde (GH) and spermine to possess amino groups were used to immobilize electrostatically the biotin labelled CSA. Exogenously added sulfated glycosaminoglycans (GAGS) [variously sulfated chondroitin sulfates and heparan sulfate (HS)] were found to compete to this immobilization in a concentration dependent mode, within a concentration range from 10 up to 300 ng/ml. Glycosaminoglycan-derived oligosaccharides competed to a degree similar to that of intact molecules. Hyaluronan (HA) and keratan sulfate (KS) did not compete the immobilization. The procedure was applied for the rapid and reproducible determination of the sulfated glycosaminoglycans in proteinase digests of small tissue samples or cell cultures with high sensitivity and accuracy.

Screening of N-acylneuraminic acids in serum and tissue specimens of mouse C57BI with Lewis' lung cancer by high-performance liquid chromatography.

Serum and tissue specimens from healthy C57BI mice and from mice with Lewis' lung cancer after metastasis were analyzed for N-acetyl- and N-glycolylneuraminic acid by high-performance liquid chromatography. Both neuraminic acids were present, while N-glycolylneuraminic acid was the predominant sialic acid in all tissues. Samples from mice with metastatic cancer showed a significant increase (67-200%) of total sialic acids mainly as a result of increased N-glycolylneuraminic acid synthesis. These results suggest that cancer metastasis in various tissues is closely associated with increased synthesis of the predominant neuraminic acid and may help to understand the underlying mechanisms of tumor development.