Using a guinea‐pig model of allergic asthma, we investigated the role of nitric oxide (NO) in allergen‐induced airway hyperreactivity after the early asthmatic reaction, by examining the effects of the NO‐synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME) on the responsiveness to methacholine and histamine of isolated perfused tracheae from unchallenged (control) animals and from animals 6 h after ovalbumin challenge. All animals developed airway hyperreactivity to inhaled histamine at 6 h after ovalbumin challenge, with a mean 3.11 ± 0.45 fold increase in sensitivity to the agonist (P < 0.001). In perfused tracheal preparations from the ovalbumin‐challenged guinea‐pigs, the maximal responses (Emax) to methacholine and histamine were significantly enhanced compared to controls, both after intraluminal (IL) and extraluminal (EL) administration of the contractile agonists. In addition, a small but significant increase in the pD2 (‐log10 EC50) for IL and EL methacholine and for IL histamine was observed. As a consequence, the ΔpD2 (EL‐IL) for histamine was slightly decreased from 1.67 ± 0.13 to 1.23 ± 0.14 (P < 0.05). However, the ΔpD2 for methacholine was unchanged (1.85 ± 0.11 and 1.77 ± 0.12, respectively; NS). Incubation of control tracheae with 100 μm L‐NAME (IL) significantly enhanced the Emax for both IL and EL methacholine and histamine to approximately the same degree as observed after ovalbumin challenge, with no effect on the pD2 and ΔpD2 for both agonists. On the contrary, L‐NAME had no effect on Emax and pD2 values of tracheal preparations from ovalbumin‐challenged guinea‐pigs. L‐NAME (10 μm‐l mM) had no effect on methacholine‐induced contraction of isolated tracheal strip preparations obtained from control animals, indicating that L‐NAME has no antimuscarinic effect on tracheal smooth muscle. Histological examination of the intact tracheal preparations indicated epithelial and subepithelial infiltration of eosinophils after ovalbumin challenge. However, no apparent damage of the airway epithelium was observed in these preparations. The results indicate that a deficiency of NO contributes to allergen‐induced airway hyperreactivity after the early asthmatic reaction and that this deficiency appears not to be due to epithelial shedding.

Background-Nitric oxide (NO) is involved in inflammation and host defence of the lung. It has been found in increased concentrations in the airways in asthmatic subjects but its levels in patients with chronic obstructive pulmonary disease (COPD) have not been investigated. A study was undertaken to determine whether markers of NO metabolism (NO in exhaled air, iNOS expression in sputum cells, and nitrite + nitrate (NO 2 -/NO 3 -) in sputum supernatant) are increased in subjects with COPD, and whether they correlate with inflammatory indices in induced sputum. The associations of these markers with smoking were also assessed. Methods-Sixteen subjects with COPD (median age 66 years, median forced expiratory volume in one second (FEV 1 ) 63% predicted, eight current smokers) and 16 healthy subjects (median age 63 years, median FEV 1 113% predicted, eight current smokers) participated in the study. NO was measured during tidal breathing and sputum was induced by inhalation of hypertonic saline. Results-No

SUMMARYTen commercially available rabbit polyclonal anti-NOS antibodies were tested for their immunohistological applicability in normal human, guinea pig, rat, and mouse organs. Most antibodies reacted as expected and described in the literature with various tissues of the investigated species. Several antibodies did not react with the expected cell populations in a certain species, or reacted in previously unknown patterns. In addition, different antibodies to the same isoform rarely detected identical cell populations, even within one species. Most of these unexpected immunoreactivities were observed in bronchial epithelial, glomerular epithelial, and vascular smooth muscle cells. These unexpected results usually occurred when the antibodies were tested in other organs or species than that to which they were originally raised. We therefore strongly recommend the use of anti-NOS antibodies only after careful immunohistological and biochemical analysis of their reactivity in the organ and species to be studied.

BackgroundCapecitabine monotherapy is a treatment option for selected patients with metastatic colorectal cancer (mCRC) and is administered to up to 17% of patients. Data are limited with regard to adverse events and dosing practices associated with capecitabine monotherapy in real-world situations.ObjectivesThe aim of this study was to provide real-world data on adverse event rates and dose adjustments/discontinuations associated with capecitabine monotherapy in patients with mCRC.MethodsThis retrospective study analyzed data from CRC patients scheduled to receive up to eight planned cycles of capecitabine monotherapy between 2009 and 2013 at a single large community hospital in The Netherlands. Data on adverse events (hand-foot syndrome [HFS], gastrointestinal (GI) events, hematological adverse events, and cardiotoxicity), as well as relative dose intensities (RDIs), dose reductions, and discontinuations, were evaluated.ResultsData from 86 patients (45 females; mean age at the start of treatment, 69 years) were included. A total of 46.5% of patients experienced HFS and 44.2% experienced a GI event at some time during treatment. Hematological events and cardiotoxicity were rare. Most patients (77%) started at below the recommended dose, and patients at the lowest dose also had the lowest median RDIs. Dose reductions and discontinuations occurred in 15–25% of patients who experienced HFS or GI event over the course of eight cycles.ConclusionsHFS and GI events were very common in patients treated with capecitabine monotherapy in a real-world clinical setting. Most patients started treatment at below the recommended dose, and 15–25% of patients who had HFS or a GI event had a dose reduction or discontinuation.

SUMMARYWe examined immunopathological changes of podocytes in vivo which, based on in vitro studies, are thought to be relevant for the pathogenesis of renal diseases. We investigated the alterations of podocytes in local inflammation in a recently developed model of pauci-immune necrotizing crescentic glomerulonephritis (NCGN) in the rat. Frozen and plastic embedded kidney sections at different time points of the disease were incubated with antibodies directed to MHC class I, MHC class II, ICAM-1 and to relevant cytokines. Strong glomerular expression of MHC class I, II and ICAM-1 was found within 4 days, and plastic embedded sections clearly demonstrated increased cell membrane staining of podocytes. Increased glomerular interferon-gamma (IFN--y) was detected within 24 h of induction of NCGN, and IL-1,3 and tumour necrosis factor-alpha (TNF-a) were found from day 4. The potency of these cytokines to induce adhesion molecules on podocytes was investigated on rat glomerular epithelial cells in vitro. By using FACS analysis and electron microscopical techniques, we found that the in vivo expression of MHC class I, II and ICAM-l by podocytes could in vitro be simulated by IFN--y. IFN-a weakly induced MHC class I, while IL-lf and TNF-a were ineffective. We hypothesize that podocytes in this in vivo model are important to maintain the local inflammatory process in the glomerulus by expression of relevant adhesion molecules and MHC molecules upon stimulation with specific cytokines. Keywords glomerular visceral epithelial cells MHC intercellular adhesion molecule-l interferon-alpha interferon

The subepithelial immune deposits of Dorus Zadel Black (DZB) rats with mercury-induced membranous nephropathy consist of autoantibodies directed to laminin P1 and of complement. The animals develop massive proteinuria within 10-14 days which is associated with obliteration of foot processes of glomerular visceral epithelial cells (GVEC), or podocytes. Previous studies indicate that these autoantibodies are probably not the sole mediator of proteinuria and GVEC damage. In this study we investigated whether circulating or macrophage-derived cytokines can contribute to the GVEC changes as detected in vivo. In vivo at the height of the proteinuria, increased intraglomerular IFN-gamma immunoreactivity was found. In diseased rats a five-fold increase in intraglomerular macrophages was found, but we could not detect intraglomerular IFN-alpha, IFN-beta, IL-1 beta or tumour necrosis factor-alpha (TNF-alpha) by using immunohistology. Subsequently, we exposed cultured GVEC to these cytokines to investigate their cytotoxic effects on several physiological and structural parameters. IFN-gamma and IL-4 were the only cytokines that exerted toxic effects, resulting in a rapidly decreased transepithelial resistance of confluent monolayers, which was closely associated with altered immunoreactivity of the tight junction protein ZO-1. IL-4 also affected vimentin and laminin immunoreactivity. IFN-gamma and IL-4 only interfered with monolayer integrity when added to the basolateral side of the GVEC, indicating specific (receptor-mediated) effects. Only IL-4 decreased the viability of the cells, and treated monolayers demonstrated an increased passage of the 44-kD protein horseradish peroxidase. From our experiments we concluded that IFN-gamma subtly affected monolayer integrity at the level of the tight junctions, and that IL-4 additionally induced cell death. We hypothesize that the toxic effects of the cytokines IFN-gamma and IL-4 as seen with cultured podocytes are necessary together with the autoantibodies, for the ultimate induction of proteinuria in mercury nephropathy in the DZB rat.

In vivo, glomerular visceral epithelial cells (GVEC), or podocytes, are morphologically highly differentiated cells which are in close contact with adjacent cells by complex interdigitating foot processes. In vitro, the dedifferentiated appearance of podocytes hampers investigations on podocyte structure and function. Cultured podocytes resemble simple epithelium in several ways with apical tight junctions and absence of foot processes. The morphological resemblances between GVEC early in embryonic development, in proteinuric diseases and in cultured cells are striking, but the mechanisms involved in these (de)differentiation processes are poorly understood. A common feature of GVEC in these various states of dedifferentiation is their altered exposure to or even total lack of hydrostatic pressure, suggesting that this may be one of the parameters involved in GVEC differentiation. In this study we investigated whether basolateral hydrostatic pressure could affect GVEC biology in vitro. We therefore exposed cultured GVEC grown on porous supports to basolateral hydrostatic pressure and investigated morphology with scanning and transmission electron microscopy, expression of specific podocyte markers and their biological responses to a model stimulus, the cytokine IFN-γ. Morphologically, monolayers of pressurized GVEC contained large regions of whirllike, raised cell formations. Individual cells in these formations had a rounded morphology and pore-like indentations between adjacent cells were observed. Cell-cell contacts were often found more basally and intercellular spaces were widened. Moreover, protein expression of pressurized monolayers was altered, as demonstrated by regions of cells with decreased keratin expression. Finally, upon exposure to the model stimulus IFN-γ, the pressurized as compared to the control GVEC demonstrated a 3-fold increased expression of MHC class II and a strongly decreased sensitivity to the toxic effects of IFN-γ. In conclusion, we found several indications that hydrostatic pressure can affect podocyte biology in vitro and similar mechanisms may account for podocyte biology in vivo. The strikingly altered morphology and biology of pressurized GVEC suggest that this culture system can be quite relevant for future studies with cultured GVEC.

There is strong evidence that vitamin D-dependent Ca(2+)-binding proteins, i.e., calbindin-D9k and calbindin-D28k, facilitate diffusion of Ca2+ through the cytosolic compartment of renal and intestinal cells, which transport Ca2+ transcellularly. In the study presented here, parvalbumin, calbindin-D9k, and calbindin-D28k were localized precisely by immunocytochemistry in rat kidney. Antisera recognizing specifically the thick ascending loop of Henle, the connecting tubules and collecting ducts, and the intercalated cells of the collecting ducts were used to identify different cell types. In rat kidney cortex, parvalbumin and calbindin-D9k colocalized in the thick ascending loop of Henle, the distal convoluted tubule, the connecting tubule, and the intercalated cells of the collecting duct. Strikingly, in all responsive cells, parvalbumin and calbindin-D9k were exclusively present in a thin layer along the basolateral membrane. In contrast, calbindin-D28k was only present in the distal convoluted and connecting tubule, where it was evenly distributed through the cytosol. In conclusion, the exclusive localization of parvalbumin and calbindin-D9k at the basolateral membrane of immunopositive renal cells implies their involvement in the regulation of transport processes located in these membranes rather than a role as intracellular Ca2+ buffer and Ca2+ shuttle between the two opposing membranes.