bluuebunny/crossref_metadata_2025 huggingface dataset:
Critical role of nuclear factor‐κB and stress‐activated protein kinases in steroid unresponsiveness
Authors: Bantel Heike , Schmitz M. Lienhard , Raible Armin , Gregor Michael , Schulze‐Osthoff Klaus
Published: October 2002

Glucocorticoid resistance is a serious clinical problem in chronic inflammatory diseases, because many patients with rheumatoid arthritis, asthma, or Crohn's disease fail to respond to steroid treatment. The molecular mechanisms underlying this unresponsiveness, however, are completely unknown. The effects of steroids are largely mediated by the interference of the glucocorticoid receptor (GR) with proinflammatory transcription factors. In the present study, we therefore investigated the activation of the transcription factors nuclear factor‐κB (NF‐κB), activator protein‐1 (AP‐1), and the upstream kinases p38 and c‐Jun N‐terminal kinase (JNK) in steroid‐sensitive and steroid‐resistant patients with Crohn's disease. We demonstrated that steroid‐sensitive and steroid‐resistant patients reveal a remarkably different cellular activation pattern of these proinflammatory mediators. In steroid‐sensitive patients, activation of NF‐κB, AP‐1, p38, and JNK was mainly found in lamina propria macrophages. In contrast, steroid‐ resistant patients revealed activation of all these mediators mostly in epithelial cells. The functional interference of the proinflammatory mediators with the glucocorticoid response was supported by reporter gene assays. Expression of NF‐κB and, interestingly, also JNK1 and p38 inhibited the activity of the GR. Thus, our results suggest that steroid resistance is associated with increased epithelial activation of stress‐activated protein kinases and NF‐κB, which might inhibit the anti‐inflammatory action of a limited number of GRs.
Decreased GFAP‐mRNA expression in spinal cord of cobalamin‐deficient rats
Authors: Magnaghi Valerio , Veber Daniela , Morabito Alberto , Buccellato Francesca R. , Melcangi Roberto C. , Scalabrino Giuseppe
Published: October 2002

We have demonstrated previously that chronic vitamin B12 [cobalamin (Cbl)] deficiency preferentially affects glial cells in the rat central nervous system (CNS) and severely affects peripheral glial cells independently of and concomitantly with the central neuropathy. In this study, we determined the mRNA levels for myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in different CNS areas of rats made Cbl‐deficient by total gastrectomy, as well as the mRNA levels for glycoprotein Po and peripheral myelin protein (PMP)22 in the sciatic nerve. GFAP‐mRNA levels were significantly decreased in the spinal cord (SC) and hypothalamus, but not in the cortex, hippocampus, or striatum of totally gastrectomized (TGX) rats. No differences in GFAP protein levels were found in the SC and hypothalamus of the TGX rats treated or not with Cbl. MBP‐mRNA levels were significantly decreased only in the hypothalamus, and the levels of mRNA for both glial markers returned to normal with Cbl replacement therapy. The levels of mRNA for the various myelin proteins in the sciatic nerve were not modified by Cbl deficiency. These results demonstrate that: a) the neurotrophic action of Cbl in rat CNS occurs in a zonal manner; and b) Cbl deficiency does not affect myelin synthesis (with the sole exception of the hypothalamus).
Vasoactive intestinal peptide and pituitary adenylate cyclase‐activating polypeptide promote in vivo generation of memory Th2 cells
Authors: Delgado Mario , Leceta Javier , Ganea Doina
Published: October 2002

Functionally active effector T cells are generated through clonal expansion. Most effector T cells are later eliminated, whereas a small number survive and differentiate into memory T cells. The mechanisms by which some effector T cells escape apoptosis and become memory T cells are not understood. Neuropeptides such as the vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase‐activating polypeptide (PACAP) inhibit antigen‐induced apoptosis of CD4 T cells. By using an in vivo long‐term experimental model, in which CD4 T cells from TRC‐transgenic mice were transferred into hosts, we demonstrate that VIP and PACAP induce the survival and/or generation of antigen‐specific CD4 T cells with a memory Th2 phenotype. This was confirmed by the fact that transgenic CD4 T cells were recovered only from mice that received Th2, but not Th1 effector cells, in the presence of VIP or PACAP. In vitro, VIP/PACAP support the survival of Th2, but not Th1, cell lines through an inhibition of antigen‐induced apoptosis. The role of neuropeptides in the biased development of Th2 memory cells is particularly relevant in view of the immune deviation existing in immune‐privileged sites such as the brain and eye, where Th2, but not Th1, responses occur in nonpathological conditions.
1α,25‐dihydroxyvitamin D3 inhibits uncoupling protein 2 expression in human adipocytes
Authors: Shi Hang , Norman Anthony W. , Okamura William H. , Sen Anindita , Zemel Michael B.
Published: October 2002

We recently demonstrated that suppressing 1α,25‐(OH)2‐D3 by increasing dietary calcium decreases adipocyte intracellular Ca2+ ([Ca2+]i), stimulates lipolysis, and inhibits lipogenesis. High calcium diets also increase core temperature and white adipose tissue uncoupling protein 2 (UCP2) expression in aP2‐agouti transgenic mice. Accordingly, we have evaluated the role of 1α,25‐(OH)2‐D3 in regulating human adipocyte UCP2 expression. Treatment of human adipocytes for 48 h with 1 nM 1α,25‐(OH)2‐D3 inhibited UCP2 mRNA and protein levels by 50% (P<0.002) and completely blocked isoproterenol‐ or fatty acid‐stimulated two‐ to threefold increases in UCP2 expression. However, a specific agonist for the membrane vitamin D receptor (mVDR), 1α,25‐dihydroxylumisterol3, was unable to inhibit basal, isoproterenol‐stimulated, or fatty acid‐stimulated UCP2 expression, whereas a specific mVDR antagonist,1β,25dihydroxyvitamin D3, was unable to prevent the 1α,25‐(OH)2‐D3 inhibition of UCP2 expression. In contrast, nuclear vitamin D receptor (nVDR) knockout via antisense oligodeoxynucleotide (ODN) prevented the inhibitory effect of 1α,25‐(OH)2‐D3 on adipocyte UCP2 expression and protein levels. These data indicate that 1α,25‐(OH)2‐D3 exerts an inhibitory effect on adipocyte UCP2 expression via the nVDR. Thus, suppression of 1α,25‐(OH)2‐D3 and consequent up‐regulation of UCP2 may contribute to our previous observation of increased thermogenesis in mice fed with high calcium diets.
Overexpression of interleukin‐15 prevents the development of murine retrovirus‐induced acquired immunodeficiency syndrome
Authors: Umemura Masayuki , Nishimura Hitoshi , Yajima Toshiki , Wajjwalk Worawid , Matsuguchi Testuya , Takahashi Masahide , Nishiyama Yukihiro , Makino Masahiko , Nagai Yoshiyuki , Yoshikai Yasunobu
Published: October 2002

LP‐BM5 murine leukemia virus (MuLV) infection causes murine acquired immunodeficiency syndrome (MAIDS), a disease characterized by varied functional abnormalities of immunocompetent cells. We found that MAIDS progression was severely retarded in IL‐15 transgenic (Tg) mice constructed with cDNA encoding secretable IL‐15 under the control of an MHC class I promoter. Several immune defects, including impaired natural killer activity, depressed IFN‐' production by T cells stimulated with anti‐T cell receptor cross‐linking, and increased susceptibility to Mycobacteium bovis infection, were prevented in IL‐15 Tg mice inoculated with LP‐BM5 MuLV. Cytotoxic T lymphocyte response to a highly antigenic 10‐mer pep‐tide encoded by LP‐BM5‐defective virus gag p12 gene was detected in the spleen and peritoneal exudate cells from IL‐15 Tg mice infected with LP‐BM5 MuLV. Intramuscular injection of cDNA encoding secretable IL‐15 also prevented the development of MAIDS. These results indicate that IL‐15 prevents the progression of MAIDS and may provide insight into an immunotherapeutic approach using the IL‐15 gene for controlling retrovirus‐induced immunodeficiency.—Umemura, M., Nishimura, H., Yajima, T., Wajjwalk, W., Matsuguchi, T., Takahashi, M., Nishiyama, Y., Makino, M., Nagai, Y., Yoshikai, Y. Overexpression of interleukin‐15 prevents the development of murine retrovirus‐induced acquired immunodeficiency syndrome. FASEB J. 16, 1755–1763 (2002)
Patterns of gene expression in atrophying skeletal muscles: response to food deprivation
Authors: Jagoe R. Thomas , Lecker Stewart H. , Gomes Marcelo , Goldberg Alfred L.
Published: October 2002

During fasting and many systemic diseases, muscle undergoes rapid loss of protein and functional capacity. To define the transcriptional changes triggering muscle atrophy and energy conservation in fasting, we used cDNA microarrays to compare mRNAs from muscles of control and food‐deprived mice. Expression of >94% of genes did not change, but interesting patterns emerged among genes that were differentially expressed: 1) mRNAs encoding polyubiquitin, ubiquitin extension proteins, and many (but not all) proteasome subunits increased, which presumably contributes to accelerated protein breakdown; 2) a dramatic increase in mRNA for the ubiquitin ligase, atrogin‐1, but not most E3s; 3) a significant suppression of mRNA for myosin binding protein H (but not other myofibrillar proteins) and IGF binding protein 5, which may favor cell protein loss; 4) decreases in mRNAs for several glycolytic enzymes and phosphorylase kinase subunits, and dramatic increases in mRNAs for pyruvate dehydrogenase kinase 4 and glutamine synthase, which should promote glucose sparing and gluconeogenesis. During fasting, metallothionein mRNA increased dramatically, mRNAs for extracellular matrix components fell, and mRNAs that may favor cap‐independent mRNA translation rose. Significant changes occurred in mRNAs for many growth‐related proteins and transcriptional regulators. These transcriptional changes indicate a complex adaptive program that should favor protein degradation and suppress glucose oxidation in muscle. Similar analysis of muscles atrophying for other causes is allowing us to identify a set of atrophy‐specific changes in gene expression.—Jagoe, R. T., Lecker, S. H., Gomes, M., Goldberg, A. L. Patterns of gene expression in atrophying skeletal muscles: response to food deprivation. FASEB J. 16, 1697–1712 (2002)
In vivo lipid‐derived free radical formation by NADPH oxidase in acute lung injury induced by lipopolysaccharide: a model for ARDS
Authors: Sato Keizo , Kadiiska Maria B. , Ghio Andrew J. , Corbett Jean , Fann Yang C. , Holland Steven M. , Thurman Ronald G. , Mason Ronald P.
Published: October 2002

Intratracheal instillation of lipopolysaccharide (LPS) activates alveolar macrophages and infiltration of neutrophils, causing lung injury/acute respiratory distress syndrome. Free radicals are a special focus as the final causative molecules in the pathogenesis of lung injury caused by LPS. Although in vitro investigation has demonstrated radical generation after exposure of cells to LPS, in vivo evidence is lacking. Using electron spin resonance (ESR) and the spin trap a‐(4‐pyridyl‐1‐oxide)‐N‐tert‐butylnitrone (POBN), we investigated in vivo free radical production by rats treated with intratracheal instillation of LPS. ESR spectroscopy of lipid extract from lungs exposed to LPS for 6 h gave a spectrum consistent with that of a POBN/ carbon‐centered radical adduct (aN=14.94±0.07 G and a:H=2.42±0.06 G) tentatively assigned as a product of lipid peroxidation. To further investigate the mechanism of LPS‐initiated free radical generation, rats were pretreated with the phagocytic toxicant GdCl3, which significantly decreased the production of radical ad‐ducts with a Corresponding decrease in neutrophil infiltration. NADPH oxidase knockout mice completely blocked phagocyte‐mediated, ESR‐detectable radical production in this model of acute lung injury. Rats treated intratracheally with LPS generate lipid‐derived free radicals via activation of NADPH oxidase.—Sato, K., Kadiiska, M. B., Ghio, A. J., Corbett, J., Fann, Y. C., Holland, S. M., Thurman, R. G., Mason, R. P. In vivo lipid‐derived free radical formation by NADPH oxidase in acute lung injury induced by lipopolysaccharide: a model for ARDS. FASEB J. 16, 1713–1720 (2002)
Insulin‐like growth factor binding protein 2 (IGFBP‐2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF‐I excess on adrenocortical cells in vivo
Authors: Hoeflich Andreas , Weber Matthias M. , Fisch Thomas , Nedbal Sabine , Fottner Christian , Elmlinger Martin W. , Wanke Rüdiger , Wolf Eckhard
Published: October 2002

GH and IGF‐I are capable of inducing cellular hypertrophy and/or hyperplasia. Chronic overexpression of GH in transgenic mice results in systemically and locally increased IGF‐I levels and in disproportionate overgrowth, including adrenocortical enlargement and corticosterone hypersecretion. Using PEPCK‐bovine GH transgenic (G) mice, we demonstrate that adrenal enlargement involves both hypertrophy (44%) and hyperplasia (50%) of zona fasciculata cells. To clarify whether IGFBP‐2 affected cell volume and number, we crossed hemizygous G mice with hemizygous CMV‐IGFBP‐2 transgenic (B) mice, generating G mice, B mice, GB double transgenic mice, and nontransgenic controls (C). The absolute weight of the adrenal glands was significantly increased in 5‐wk‐and 4‐month‐old G mice vs. C and B mice. IGFBP‐2 overexpression in GB mice reduced this effect of GH excess by 26% and 37% in 5‐wk‐and 4‐month‐old animals, respectively. GH‐induced hypertrophy of zona fasciculata cells was completely abolished by IGFBP‐2 overexpression in GB mice whereas hyperplasia was not affected. Basal and ACTH‐induced plasma corticosterone levels of 4‐month‐old G mice, but not of GB mice, were two‐to threefold increased compared with C mice. Plasma ACTH levels were similar in all groups. Our data show that IGFBP‐2 potently separates hypertrophic and hyperplastic effects of GH/IGF‐I excess on adrenocortical cells.—Hoeflich, A., Weber, M. M., Fisch, T., Nedbal, S., Fottner, C., Elmlinger, M. W., Wanke, R., Wolf, E. Insulin‐like growth factor binding protein 2 (IGFBP‐2) separates hypertrophic and hyperplastic effects of growth hormone (GH)/IGF‐I excess on adrenocortical cells in vivo. FASEB J. 16, 1721–1731 (2002)
Concomitant S‐, N‐, and heme‐nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo
Authors: Feelisch Martin , Rassaf Tienush , Mnaimneh Sanie , Singh Nisha , Bryan Nathan S. , Jourd'Heuil David , Kelm Malte
Published: October 2002

There is growing evidence for the involvement of nitric oxide (NO) ‐mediated nitrosation in cell signaling and pathology. Although S‐nitrosothiols (RSNOs) have been frequently implicated in these processes, it is unclear whether NO forms nitrosyl adducts with moieties other than thiols. A major obstacle in assessing the significance of formation of nitro‐sated species is the limited reliability of available analytical techniques for measurements in complex biological matrices. Here we report on the presence of nitrosated compounds in plasma and erythrocytes of rats, mice, guinea pigs, and monkeys under basal conditions, in immunologically challenged murine macrophages in vitro and laboratory animals in vivo. Besides RSNOs, all biological samples also contained mercury‐stable nitroso species, indicating the additional involvement of amine and heme nitros(yl)ation reactions. Significant differences in the amounts and ratios of RSNOs over N‐and heme‐nitros(yl)ated compounds were found between species and organs. These observations were made possible by the development of a novel gas‐phase chemiluminescence‐based technique that allows detection of nitroso species in tissues and biological fluids without prior extraction or deproteinization. The method can quantify as little as 100 fmol bound NO and has been validated extensively for use in different biological matrices. Discrimination between nitrite, RSNOs, and N‐nitroso or nitrosylheme compounds is accomplished by use of group‐specific reagents. Our findings suggest that NO generation in vivo leads to concomitant formation of RSNOs, nitrosamines, and nitrosylhemes with considerable variation between rodents and primates, highlighting the difficulty in comparing data between different animal models and extrapolating results from experimental animals to human physiology.—Feelisch, M., Rassaf, T., Mnaimneh, S., Singh, N., Bryan, N. S., Jourd'heuil, D., Kelm, M. Concomitant S‐, N‐, and heme‐nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J. 16, 1775–1785 (2002)
Prevention of spontaneous mammary adenocarcinoma in HER‐2/neu transgenic mice by foreign DNA
Authors: Sfondrini Lucia , Besusso Dario , Rumio Cristiano , Rodolfo Monica , Ménard Sylvie , Balsari Andrea
Published: October 2002

Unmethylated CpG‐oligodeoxynucleotides (CpG‐ODNs) are recognized as a ‘danger signal’ and are potent immunostimulators. To test whether tumors might be prevented by maintaining the innate immune system on continuous alert, proto‐neu transgenic female mice, which develop spontaneous mammary tumors, were systemically treated with CpG‐ODNs at 10‐day intervals. Tumor incidence and number of tumors/mouse were significantly lower in treated mice compared with the control group. Moreover, CpG‐ODN systemic treatment significantly reduced lung metastases induced by intravenous inoculation of N202.1A cells derived from a spontaneous mammary carcinoma. Growth of established tumors was modestly inhibited after CpG‐ODN systemic treatment but strongly on peritumoral application. Our data indicate that systemic repeated injection of CpG‐ODN to maintain the innate immune system on continuous alert prevents the onset of genetically determined tumors and confers tumor protection when the tumor load is low.—Sfondrini, L., Besusso, D., Rumio, C., Rodolfo, M., Ménard, S., Balsari, A. Prevention of spontaneous mammary adenocarcinoma in HER‐2/neu transgenic mice by foreign DNA. FASEB J. 16, 1749–1754 (2002)
Cell culture on thin tissue sections commonly prepared for histopathology
Authors: Takezawa Toshiaki , Takenouchi Takato , Imai Kei , Takahashi Toru , Hashizume Kazuyoshi
Published: October 2002

Thin tissue sections commonly prepared on a glass slide for histopathology retain many in vivo biochemical attributes related not only to structure but also function. We hypothesized that such tissue sections might serve as novel cell culture substrata that would reflect tissue conditions in vivo. Here we report the applicability of tissue section substrata to tissue reconstruction and serum‐free culture. Four different cell types were cultured on section and acellularized section substrata prepared from a bovine placenta. The labyrinth region of the substratum induced cell differentiation to elicit the formation of multicellular spheroids of BeWo cells (human choriocarcinoma cells), a capillary network‐like structure for CPAE cells (bovine pulmonary artery endothelial cells), and a neuronal network‐like structure for PC‐12 cells (rat pheochromocytoma cells). The substratum provided a microenvironment that maintained the viability of PC‐12 cells in a serum‐free culture. We also succeeded in preparing a multicellular mass of normal human dermal fibroblasts (NHDFs) involving acellularized section‐derived components. This technology offers the novel investigation of cell behaviors induced by culturing different cell types on various tissue sections and will be a useful tool for identifying cell characteristics and clarifying the molecular mechanisms that regulate the behavior of each cell type.
Glyceroneogenesis comes of age
Authors: Beale Elmus G. , Hammer Robert E. , Antoine Bénédicte , Forest Claude
Published: October 2002

Glyceroneogenesis is a generally ignored metabolic pathway that occurs in adipose tissues and liver of mammalian species. This short review highlights a series of recent discoveries showing that glyceroneogenesis is important in lipid homeostasis.—Beale, E. G., Hammer, R. E., Antoine, B., Forest, C. Glyceroneogenesis comes of age. FASEB J. 16, 1695–1696 (2002)
Lipoxin, leukotriene, and PDGF receptors cross‐talk to regulate mesangial cell proliferation
Authors: Mc Mahon Blaithin , Mitchell Derick , Shattock Richard , Martin Finian , Brady Hugh R. , Godson Catherine
Published: October 2002

The lipoxygenase‐derived leukotrienes (LTs) are important proinflammatory lipid mediators. Lipoxins (LXs), more recently described lipoxygenase products, modulate many proinflammatory actions of LTs and have impressive proresolution properties. Mesangial cell (MC) proliferation is a central event in the pathogenesis of glomerulonephritis. LTD4‐induced proliferation of mesangial cells is modulated by LXA4. Here, we demonstrate that LXA4 inhibits PDGF‐ and LTD4‐stimulated proliferation through modulation of platelet‐derived growth factor receptor β (PDGFRβ) activation. Specifically, we demonstrate that LTD4 transactivates the PDGFRβ, a process associated with c‐src recruitment and ras activation. We demonstrate expression of cysLT1 and cysLT2 receptors in MCs. LTD4‐induced c‐src activation was insensitive to pertussis toxin and the cysLT1 receptor antagonist Zafirlukast but was blocked by the nonselective antagonist Pobilukast. We show that LXA4 inhibits LTD4‐stimulated activation of the PDGFRβ and that LXA4 modulates PDGF‐BB‐stimulated tyrosine phosphorylation of the PDGFRβ and subsequent mitogenic events. Furthermore, expression of recombinant LXA4 receptor (ALXR) in CHOK1 cells was associated with an attenuation of serum‐stimulated proliferation. These data demonstrate that LXA4 receptor (ALXR) activation is accompanied by antimitogenic effects coupled with inactivation of growth factor receptors, highlighting the complex cross‐talk between G protein‐coupled receptors and receptor tyrosine kinases in an inflammatory milieu. These data elaborate on the profile of cell signaling events that underpin the anti‐inflammatory and proresolution bioactions of LX.
TNF‐α signal transduction in rat neonatal cardiac myocytes: definition of pathways generating from the TNF‐α receptor
Authors: Condorelli Gianluigi , Morisco Carmine , Latronico Michael V. G. , Claudio Pier Paolo , Dent Paul , Tsichlis Philip , Condorelli Gerolama , Frati Giacomo , Drusco Alessandra , Croce Carlo M. , Napoli Claudio
Published: October 2002

Cardiomyocyte hypertrophy and apoptosis have been implicated in the loss of contractile function during heart failure (HF). Moreover, patients with HF have been shown to exhibit increased levels of tumor necrosis factor a (TNF‐a) in the myocardium. However, the multiple signal transduction pathways generating from the TNF‐α receptor in cardiomyocytes and leading preferentially to apoptosis or hypertrophy are still unknown. Here we demonstrate in neonatal rat cardiomyocytes that 1) TNF‐α induces phosphorylation of AKT, activation of NF‐κB, and the phosphorylation of JUN kinase; 2) blocking AKT activity prevents NF‐κB activation, suggesting a role for AKT in regulating NF‐κB function; 3) AKT and JUN are both critical for the hypertrophic effects of TNF‐α, since dominant‐negative mutants of these genes are capable of inhibiting TNF‐α‐induced ANF‐promoter up‐regulation and increase in cardiomyocyte cell size, and 4) blocking NF‐κB, AKT, or JUN alone or in combination does not sensitize cardiomyocytes to the proapoptotic effects of TNF‐α, in contrast to other cell types, suggesting a cardiac‐specific pathway regulating the anti‐apoptotic events induced by TNF‐α. Altogether, the data presented evidence the role of AKT and JUN in TNF‐αinduced cardiomyocyte hypertrophy and apoptosis.—Condorelli, G., Morisco, C., Latronico, M., Claudio, P. P., Dent, P., Tsichlis, P., Condorelli, G., Frati, G., Drusco, A., Croce, C. M., Napoli, C. TNF‐α signal transduction in rat neonatal cardiac myocytes: definition of pathways generating from the TNF‐α receptor. FASEB J. 16, 1732–1737 (2002)
Loss of sarcolemma nNOS in sarcoglycan‐deficient muscle
Authors: Crosbie Rachelle H. , Barresi Rita , Campbell Kevin P.
Published: October 2002

nNOS, anchored to the sarcolemma through its interactions with the dystrophin–glycoprotein complex, is dramatically reduced in dystrophindeficient mdx mice and Duchenne muscular dystrophy patients. Recent evidence suggests that loss of nNOS in dystrophin‐deficient muscle may contribute significantly to the progression of muscle pathology through a variety of mechanisms. To investigate whether nNOS plays a role in other forms of muscular dystrophy, we analyzed protein expression of nNOS in several sarcoglycan‐deficient animal models of muscular dystrophy as well as patients with primary mutations in the sarcoglycan genes. Primary mutations in α‐, β‐, δ‐, and γ‐sarcoglycan result in autosomal recessive limb girdle muscular dystrophy (AR‐LGMD). We report that loss of the sarcoglycan–sarcospan complex in muscle causes a dramatic reduction in the levels of nNOS expression at the membrane, even in the presence of normal dystrophin and syntrophin expression. Furthermore, we show that expression of three out of four sarcoglycans is not sufficient to maintain nNOS at the sarcolemma. Our data suggest that loss of nNOS may contribute to muscle pathology in AR‐LGMD with primary mutations in the sarcoglycans.—Crosbie, R. H., Barresi, R., Campbell, K. P. Loss of sarcolemma nNOS in sarcoglycandeficient muscle. FASEB J. 16, 1786–1791 (2002)
Interaction of pre‐programmed control and natural stretch reflexes in human landing movements
Authors: McDonagh Martin J. N. , Duncan Audrey
Published: November 2002

Pre‐programmed mechanisms of motor control are known to influence the gain of artificially evoked stretch reflexes. However, their interaction with stretch reflexes evoked in the context of unimpeded natural movement is not understood. We used a landing movement, for which a stretch reflex is an integral part of the natural action, to test the hypothesis that unpredicted motor events increase stretch reflex gain. The unpredicted event occurred when a false floor, perceived to be solid, collapsed easily on impact, allowing the subjects to descend for a further 85 ms to a solid floor below. Spinal stretch reflexes were measured following solid floor contact. When subjects passed through the false floor en route to the solid floor, the amplitude of the EMG reflex activity was double that found in direct falls. This was not due to differences in joint rotations between these conditions. Descending pathways can modify H‐ and stretch‐reflex gain in man. We therefore manipulated the time between the false and real floor contacts and hence the time available for transmission along these pathways. With 30 ms between floors, the enhancement of the reflex was extinguished, whereas with 50 ms between floors it reappeared. This excluded several mechanisms from being responsible for the doubling of the reflex EMG amplitude. It is argued that the enhanced response is due to the modulation of reflex gain at the spinal level by signals in descending pathways triggered by the false platform. The results suggest the future hypothesis that this trigger could be the absence of afferent signals expected at the time of false floor impact and that salient error signals produced from a comparison of expected and actual sensory events may be used to reset reflex gains.
Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay for Detection and Differentiation of Antibodies against European and North American Porcine Reproductive and Respiratory Syndrome Virus
Authors: Seuberlich Torsten , Tratschin Jon-Duri , Thür Barbara , Hofmann Martin A.
Published: November 2002

Two types of porcine reproductive and respiratory syndrome virus (PRRSV) have been reported, the European type (EU PRRSV) and the North American type (US PRRSV). We developed a dual enzyme-linked immunosorbent assay (ELISA) for the simultaneous detection and differentiation of serum antibodies directed against either of the two PRRSV types. This tandem PRRS ELISA is based on affinity-purified recombinant nucleocapsid protein expressed in
Escherichia coli
. Sensitivity and specificity were assessed by using the IDEXX HerdChek PRRS ELISA and the indirect immunofluorescence assay as reference tests. A total of 1,571 sera originating from the United States, Europe, and two PRRS-free countries, i.e., Switzerland and New Zealand, were used for validation of the tandem PRRS ELISA. The new test performed at least as well as the reference tests in regard to sensitivity (0.94 for the US PRRS ELISA and 0.93 for the EU PRRS ELISA) and specificity (0.96 for the US PRRS ELISA and 0.99 for the EU PRRS ELISA). Positive sera were correctly differentiated in 582 of 591 cases, indicating a high differentiation capability of this dual ELISA. The robustness and repeatability of the test were assessed and found to be appropriate for diagnostic applications. Taken together, the data indicate that the tandem PRRS ELISA described here is the first differentiation ELISA for PRRSV serology based on recombinant antigen. It is convenient with respect to antigen production, and it is reliable, economical, and highly sensitive and specific. Thus, it is considered to be a powerful tool for routine diagnostics, epidemiological surveys, and outbreak investigations.
Tumor Necrosis Factor Gene Polymorphisms, Leukocyte Function, and Sepsis Susceptibility in Blunt Trauma Patients
Authors: Majetschak Matthias , Obertacke Udo , Schade F. Ulrich , Bardenheuer Mark , Voggenreiter Gregor , Bloemeke Brunhilde , Heesen Michael
Published: November 2002

The tumor necrosis factor alpha (TNF-α) −308 G/A and TNF-β NcO1 polymorphisms have been described to be associated with an increased risk for sepsis in critically ill patients. Functional consequences associated with these polymorphisms remain unclear. We compared the genotype distribution of these TNF polymorphisms with susceptibility to severe sepsis and leukocyte function in blunt trauma patients (n= 70; mean injury severity score, 24 points [range, 4 to 57). Severe sepsis was defined according to the American College of Chest Physicians-Society of Critical Care Medicine consensus conference criteria. Genotyping for the NcO1 polymorphism (alleles TNFB1 and TNFB2) was performed by PCR and digestion of the products with NcO1, and that for the TNF-α −308 G/A polymorphism (alleles TNF1 and TNF2) was performed by real-time PCR. Leukocyte function was assessed by measurement of the production of endotoxin-induced cytokines (TNF-α, interleukin-6 [IL-6], and IL-8) in whole blood. TNF-α, IL-6, and IL-8 were determined by enzyme-linked immunosorbent assay. For the genotypes of the TNF-α −308 G/A polymorphism, differences in the frequency of development of severe sepsis were not detectable. Patients developing severe sepsis after trauma were significantly more likely to posses a homozygous genotype of the TNF-β NcO1 polymorphism. Compared with heterozygotes, the odds ratio for the TNFB2/B2 genotype for the development of severe posttraumatic sepsis was 11 (P= 0.01), and that for the TNFB1/B1 genotype was 13 (P= 0.014). TNF-α −308:TNF-β NcO1 haplotype analysis showed that the TNFB2:TNF2 haplotype is significantly negatively associated with development of severe sepsis. Patients homozygous for the TNFB1 or TNFB2 allele showed a persistently higher cytokine-producing capacity during at least 4 to 8 days after trauma than the heterozygotes. In patients homozygous for the TNF1 allele, a higher TNF-α- and IL-8-producing capacity was found only at day 1 after trauma. Although the TNF-β NcO1 polymorphism appears to be less likely to be causative for development of severe sepsis after trauma, it is thus far the only genetic marker identified which can be used as a relevant risk estimate for severe sepsis in trauma patients immediately after the injury.
Streptokinase Promotes Development of Dipeptidyl Peptidase IV (CD26) Autoantibodies after Fibrinolytic Therapy in Myocardial Infarction Patients
Authors: Cuchacovich Miguel , Gatica Héctor , Vial Paula , Yovanovich Jorge , Pizzo Salvatore V. , Gonzalez-Gronow Mario
Published: November 2002

Dipeptidyl peptidase IV (DPP IV) (CD26) plays a critical role in the modulation and expression of autoimmune and inflammatory diseases. We recently reported that sera from patients with rheumatoid arthritis and systemic lupus erythematosus contained low levels of DPP IV and high titers of anti-DPP IV autoantibodies of the immunoglobulin A (IgA) and IgG classes and found a correlation between the low circulating levels of DPP IV and the high titers of anti-DPP IV autoantibodies of the IgA class. Since streptokinase (SK) is a potent immunogen and binds to DPP IV, we speculated that patients with autoimmune diseases showed higher DPP IV autoantibody levels than healthy controls as a consequence of an abnormal immune stimulation triggered by SK released during streptococcal infections. We assessed this hypothesis in a group of patients suffering from acute myocardial infarction, without a chronic autoimmune disease, who received SK as part of therapeutic thrombolysis. Concomitant with the appearance of anti-SK antibodies, these patients developed anti-DPP IV autoantibodies. These autoantibodies bind to DPP IV in the region which is also recognized by SK, suggesting that an SK-induced immune response is responsible for the appearance of DPP IV autoantibodies. Furthermore, we determined a correlation between high titers of DPP IV autoantibodies and an augmented clearance of the enzyme from the circulation. Serum levels of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin 6 (IL-6) increased significantly after 30 days of SK administration, while the levels of soluble IL-2 receptor remained unchanged during the same period, suggesting a correlation between the lower levels of circulating DPP IV and higher levels of TNF-α and IL-6 in serum in these patients.
Leishmania major-Like Antigen for Specific and Sensitive Serodiagnosis of Human and Canine Visceral Leishmaniasis
Authors: Barbosa-de-Deus Rosângela , dos Mares-Guia Marcos Luíz , Nunes Adriane Zacarias , Costa Kátia Morais , Junqueira Roberto Gonçalves , Mayrink Wilson , Genaro Odair , Tavares Carlos Alberto Pereira
Published: November 2002

An antigen (LMS) prepared fromLeishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1,822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas' disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas' disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested.
Comparison ofStaphylococcus aureusIsolates from Bovine and Human Skin, Milking Equipment, and Bovine Milk by Phage Typing, Pulsed-Field Gel Electrophoresis, and Binary Typing
Authors: Zadoks R. N. , van Leeuwen W. B. , Kreft D. , Fox L. K. , Barkema H. W. , Schukken Y. H. , van Belkum A.
Published: October 2002

Staphylococcus aureusisolates (n= 225) from bovine teat skin, human skin, milking equipment, and bovine milk were fingerprinted by pulsed-field gel electrophoresis (PFGE). Strains were compared to assess the role of skin and milking equipment as sources ofS. aureusmastitis. PFGE ofSmaI-digested genomic DNA identified 24 main types and 17 subtypes among isolates from 43 herds and discriminated between isolates from bovine teat skin and milk. Earlier, phage typing (L. K. Fox, M. Gershmann, D. D. Hancock, and C. T. Hutton, Cornell Vet. 81:183-193, 1991) had failed to discriminate between isolates from skin and milk. Skin isolates from humans belonged to the same pulsotypes as skin isolates from cows. Milking equipment harbored strains from skin as well as strains from milk. We conclude thatS. aureusstrains from skin and from milk can both be transmitted via the milking machine, but that skin strains are not an important source of intramammaryS. aureusinfections in dairy cows. A subset of 142 isolates was characterized by binary typing with DNA probes developed for typing of humanS. aureus. Typeability and overall concordance with epidemiological data were lower for binary typing than for PFGE while discriminatory powers were similar. Within several PFGE types, binary typing discriminated between main types and subtypes and between isolates from different herds or sources. Thus, binary typing is not suitable as replacement for PFGE but may be useful in combination with PFGE to refine strain differentiation.
Molecular Epidemiology of Pneumococcal Carriage among Children with Upper Respiratory Tract Infections in Hanoi, Vietnam
Authors: Bogaert D. , Ha N. T. , Sluijter M. , Lemmens N. , de Groot R. , Hermans P. W. M.
Published: October 2002

To investigate the molecular epidemiology of pneumococcal nasopharyngeal carriage in Hanoi, Vietnam, we studied 84 pneumococcal strains retrieved from children with upper respiratory tract infections. Serotypes 23F (32%), 19F (21%), 6B (13%), and 14 (10%) were found most often. A significant number of strains were antibiotic resistant. Fifty-two percent of the strains were (intermediate) resistant to penicillin, 87% were (intermediate) resistant to co-trimoxazole, 76% were resistant to tetracycline, 73% were resistant to erythromycin, and 39% were (intermediate) resistant to cefotaxime. Seventy-five percent were resistant to three or more classes of antibiotics. A high degree of genetic heterogeneity among the penicillin resistance genes was observed. In addition, the tetracycline resistance gene
tet
(M) and the erythromycin resistance gene
erm
(B) were predominantly observed among the isolates. Molecular analysis of the 84 isolates by restriction fragment end labeling (RFEL) revealed 35 distinct genotypes. Twelve of these genotypes represented a total of eight genetic clusters with 61 isolates (73%). The two largest clusters contained 24 and 12 isolates, and the isolates in those clusters were identical to the two internationally spreading multidrug-resistant clones Spain 23F-1 and Taiwan 19F-14, respectively. The remaining RFEL types were Vietnam specific, as they did not match the types in our reference collection of 193 distinct RFEL types from 16 countries. Furthermore, 57 of the 61 horizontally spreading isolates (93%) in the eight genetic clusters were covered by the seven-valent conjugate vaccine, whereas this vaccine covered only 43% of the isolates with unique genotypes. According to the serotype distribution of the nasopharyngeal pneumococcal isolates, this study suggests a high potential benefit of the seven-valent pneumococcal conjugate vaccine for children in Hanoi.
Increased Risk of Parvovirus B19 Infection in Young Adult Cancer Patients Receiving Multiple Courses of Chemotherapy
Authors: Kuo Sung-Hsin , Lin Liang-In , Chang Chee-Jen , Liu Yun-Ru , Lin Kuo-Sin , Cheng Ann-Lii
Published: October 2002

An increased human parvovirus B19 infection rate has been observed in immunocompromised hosts. In this study, we sought to determine the prevalence of parvovirus B19 infection in adult cancer patients receiving multiple courses of systemic chemotherapy. From March 1999 through April 2000, 59 men and 68 women, with a median age of 49 (18 to 79) years, were enrolled in this study. They had received an average of 7.1 (4 to 32) courses of systemic chemotherapy. The median duration from the date of starting chemotherapy to the date of blood sampling was 11 (4 to 88) months. Serum B19 immunoglobulin G (IgG) and IgM levels were examined by an enzyme-linked immunosorbent assay, and B19 DNA was examined by a nested PCR. A group of 400 healthy blood donors served as the control group. The overall prevalences of anti-B19 IgG in adult cancer patients and healthy blood donors were 61.4 and 25.0%, respectively (P< 0.01). Anti-B19 IgM and B19 DNA were not detectable in these anti-B19 IgG-seropositive individuals. A further age-stratified comparison revealed that only patients younger than 40 years had a significantly higher anti-B19 IgG seropositivity rate than the controls (19 of 39 versus 53 of 310;P< 0.001). The increased prevalence of B19 infection in these 39 adult patients younger than 40 years might be clinically significant, since unexplained anemia, a common sequela of B19 infection, was detected in 3 of 20 seronegative patients (15.0%) and in 12 of 19 seropositive patients (63.2%) (P< 0.005). The results of this study suggest that adult patients younger than 40 years and receiving multiple courses of systemic chemotherapy may have a significantly increased risk of B19 infection. Prospective studies to define the time course and clinical consequence of B19 infection in this group of patients are needed.
Alafosfalin as a Selective Agent for Isolation of
Salmonella
from Clinical Samples
Authors: Perry J. D. , Riley G. , Gould F. K. , Perez J. M. , Boissier E. , Ouedraogo R. T. , Freydière A. M.
Published: October 2002

The selectivity of a range of culture media for the detection of
Salmonella
was assessed using 435 strains of gram-negative bacteria. These media showed limited ability to inhibit non-
Salmonella
strains found in stool samples. We report the evaluation of alafosfalin as a selective agent for isolation of
Salmonella
from stool samples. Susceptibility studies with this agent showed that non-
typhi Salmonella
strains were relatively resistant (mean MIC, 10.2 mg/liter) compared to many coliforms including
Escherichia coli
(mean MIC, 0.7 mg/liter). A chromogenic medium, ABC medium, was modified to incorporate alafosfalin and was compared with standard ABC medium and Hektoen enteric agar for the isolation of
Salmonella
from 1,000 stool samples. On direct culture, modified ABC medium showed higher recovery of
Salmonella
(53.6%) compared with either ABC medium (35.7%) or Hektoen enteric agar (48.2%). We conclude that alafosfalin is a useful selective agent for the isolation of
Salmonella
from stool samples.
Rates of Detection of
Neisseria meningitidis
in Tonsils Differ in Relation to Local Incidence of Invasive Disease
Authors: Greiner Oliver , Berger Christoph , Day Philip J. R. , Meier Gabriela , Tang Christoph M. , Nadal David
Published: October 2002

Nasopharyngeal swabbing substantially underestimates carriage of
Neisseria meningitidis
. Real-time PCR assays were employed to examine the presence of a broad range of bacteria and of
N. meningitidis
groups B and C, respectively, in tonsils from 26 individuals from Oxford, England, and 72 individuals from Zurich, Switzerland. The detection limit of each PCR system was DNA from one bacterial cell per reaction mixture. Tonsillar DNA did not inhibit amplification of meningococcal gene sequences, and
N. meningitidis
was detected in tonsils exposed to the bacterium. Whereas in both sets of patients other bacteria were detected,
N. meningitidis
group B and group C were only found in tonsils from Oxford where the incidence of invasive meningococcal disease is much higher than in Zurich. These observations suggest that PCR-based methods could be used for the detection of meningococcal carriage and that difference in disease incidence could be explained by different transmission rates in the community rather than host genetics or coexisting infections.
Real-Time PCR Method for Detection ofEncephalitozoonintestinalisfrom Stool Specimens
Authors: Wolk D. M. , Schneider S. K. , Wengenack N. L. , Sloan L. M. , Rosenblatt J. E.
Published: October 2002

The prevalence of microsporidiosis is likely underestimated due to the labor-intensive, insensitive, and nonspecific clinical laboratory methods used for the diagnosis of this disease. A real-time PCR assay was designed to assess DNA extraction methods and to detect threeEncephalitozoonspecies in feces. Modifications of the MagNA Pure LC DNA isolation kit protocol (Roche Applied Sciences, Indianapolis, Ind.) were compared by using the automated MagNA Pure LC instrument (Roche) and fecal specimens spiked withEncephalitozoonintestinalisspores. Extracted DNA was amplified by the LightCycler (Roche) PCR assay. Assay sensitivity, reproducibility, and efficiency were assessed by comparing threshold crossover values achieved with different extraction and storage conditions (fresh, refrigerated, frozen, and preserved specimens). Optimal extraction conditions were achieved by using a commercial buffer, tissue lysis buffer (Roche), as the specimen diluent. LightCycler PCR results were compared to those obtained from routine stool microscopy with trichrome blue stain. The lower limit of detection for the LightCycler PCR assay varied by storage conditions from 102to 104spores/ml of feces, a value which represented a significant improvement over that achieved by staining (≥1.0 × 106spores/ml). Melting temperature analysis of the amplicons allowed for the differentiation of threeEncephalitozoonspecies (E.intestinalis,E.cuniculi, andE.hellem). The assay is readily adaptable to the clinical laboratory and represents the first real-time PCR assay designed to detectEncephalitozoonspecies.
Validation of Performance of the Gen-Probe Human Immunodeficiency Virus Type 1 Viral Load Assay with Genital Swabs and Breast Milk Samples
Authors: DeVange Panteleeff Dana , Emery Sandra , Richardson Barbra A. , Rousseau Christine , Benki Sarah , Bodrug Sharon , Kreiss Joan K. , Overbaugh Julie
Published: October 2002

Human immunodeficiency type 1 (HIV-1) continues to spread at an alarming rate. The virus may be transmitted through blood, genital secretions, and breast milk, and higher levels of systemic virus in the index case, as measured by plasma RNA viral load, have been shown to correlate with increased risk of transmitting HIV-1 both vertically and sexually. Less is known about the correlation between transmission and HIV-1 levels in breast milk or genital secretions, in part because reliable quantitative assays to detect HIV-1 in these fluids are not available. Here we show that the Gen-Probe HIV-1 viral load assay can be used to accurately quantify viral load in expressed breast milk and in cervical and vaginal samples collected on swabs. Virus could be quantified from breast milk and swab samples spiked with known amounts of virus, including HIV-1 subtypes A, C, and D. As few as 10 copies of HIV-1 RNA could be detected above background threshold levels in ≥77% of assays performed with spiked breast milk supernatants and mock swabs. In genital swab samples from HIV-1-infected women, similar levels of HIV-1 RNA were consistently detected in duplicate swabs taken from the same woman on the same clinic visit, suggesting that the RNA values from a single swab sample can be used to measure genital viral load.