Autophagy is a novel pathway for neurofilament protein degradation in vivo.

How macroautophagy/autophagy influences neurofilament (NF) proteins in neurons, a frequent target in neurodegenerative diseases and injury, is not known. NFs in axons have exceptionally long half-lives in vivo enabling formation of large stable supporting networks, but they can be rapidly degraded during Wallerian degeneration initiated by a limited calpain cleavage. Here, we identify autophagy as a previously unrecognized pathway for NF subunit protein degradation that modulates constitutive and inducible NF turnover in vivo. Levels of NEFL/NF-L, NEFM/NF-M, and NEFH/NF-H subunits rise substantially in neuroblastoma (N2a) cells after blocking autophagy either with the phosphatidylinositol 3-kinase (PtdIns3K) inhibitor 3-methyladenine (3-MA), by depleting ATG5 expression with shRNA, or by using both treatments. In contrast, activating autophagy with rapamycin significantly lowers NF levels in N2a cells. In the mouse brain, NF subunit levels increase in vivo after intracerebroventricular infusion of 3-MA. Furthermore, using tomographic confocal microscopy, immunoelectron microscopy, and biochemical fractionation, we demonstrate the presence of NF proteins intra-lumenally within autophagosomes (APs), autolysosomes (ALs), and lysosomes (LYs). Our findings establish a prominent role for autophagy in NF proteolysis. Autophagy may regulate axon cytoskeleton size and responses of the NF cytoskeleton to injury and disease.

Neuroprotection mediated by cystatin C-loaded extracellular vesicles.

Cystatin C (CysC) is implicated in neuroprotection and repair in the nervous system in response to diverse neurotoxic conditions. In addition to being secreted from cells in a soluble form, CysC is released by cells in association with extracellular vesicles (EVs), including exosomes. We demonstrate that EVs containing CysC protect cultured cells from starvation-induced death. Moreover, while EVs secreted by CysC-deficient cells were not protective, EVs secreted by CysC-deficient cells treated with exogenous human CysC significantly enhanced the survival of the cells. CysC also plays a role in modulating the secretion of EVs, enhancing secretion of EVs by primary cortical neurons and primary cortical smooth muscle cells. Confirming these in vitro findings, higher EV levels were observed in the brain extracellular space of transgenic mice expressing human CysC as compared to littermate controls. Regulation of cell-secreted EV levels and content in the brain is likely to be essential to maintaining normal brain function. We propose that enhanced EV release could rescue the deleterious effects of dysfunction of the endosomal-lysosomal system in neurodegenerative disorders. Moreover, a higher level of CysC-loaded EVs released from cells in the central nervous system has important protective functions, representing a potential therapeutic tool for disorders of the central nervous system.

Cyclodextrin has conflicting actions on autophagy flux in vivo in brains of normal and Alzheimer model mice.

2-hydroxypropyl-ß-cyclodextrin (CYCLO), a modifier of cholesterol efflux from cellular membrane and endo-lysosomal compartments, reduces lysosomal lipid accumulations and has therapeutic effects in animal models of Niemann-Pick disease type C and several other neurodegenerative states. Here, we investigated CYCLO effects on autophagy in wild-type mice and TgCRND8 mice-an Alzheimer's Disease (AD) model exhibiting ß-amyloidosis, neuronal autophagy deficits leading to protein and lipid accumulation within greatly enlarged autolysosomes. A 14-day intracerebroventricular administration of CYCLO to 8-month-old TgCRND8 mice that exhibit moderately advanced neuropathology markedly diminished the sizes of enlarged autolysosomes and lowered their content of GM2 ganglioside and Aß-immunoreactivity without detectably altering amyloid precursor protein processing or extracellular Aß/ß-amyloid burden. We identified two major actions of CYCLO on autophagy underlying amelioration of lysosomal pathology. First, CYCLO stimulated lysosomal proteolytic activity by increasing cathepsin D activity, levels of cathepsins B and D and two proteins known to interact with cathepsin D, NPC1 and ABCA1. Second, CYCLO impeded autophagosome-lysosome fusion as evidenced by the accumulation of LC3, SQSTM1/p62, and ubiquitinated substrates in an expanded population of autophagosomes in the absence of greater autophagy induction. By slowing substrate delivery to lysosomes, autophagosome maturational delay, as further confirmed by our in vitro studies, may relieve lysosomal stress due to accumulated substrates. These findings provide in vivo evidence for lysosomal enhancing properties of CYCLO, but caution that prolonged interference with cellular membrane fusion/autophagosome maturation could have unfavorable consequences, which might require careful optimization of dosage and dosing schedules.

Calpastatin inhibits motor neuron death and increases survival of hSOD1(G93A) mice.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron disease with a poorly understood cause and no effective treatment. Given that calpains mediate neurodegeneration in other pathological states and are abnormally activated in ALS, we investigated the possible ameliorative effects of inhibiting calpain over-activation in hSOD1(G93A) transgenic (Tg) mice in vivo by neuron-specific over-expression of calpastatin (CAST), the highly selective endogenous inhibitor of calpains. Our data indicate that over-expression of CAST in hSOD1(G93A) mice, which lowered calpain activation to levels comparable to wild-type mice, inhibited the abnormal breakdown of cytoskeletal proteins (spectrin, MAP2 and neurofilaments), and ameliorated motor axon loss. Disease onset in hSOD1(G93A) /CAST mice compared to littermate hSOD1(G93A) mice is delayed, which accounts for their longer time of survival. We also find that neuronal over-expression of CAST in hSOD1(G93A) transgenic mice inhibited production of putative neurotoxic caspase-cleaved tau and activation of Cdk5, which have been implicated in neurodegeneration in ALS models, and also reduced the formation of SOD1 oligomers. Our data indicate that inhibition of calpain with CAST is neuroprotective in an ALS mouse model. CAST (encoding calpastatin) inhibits hyperactivated calpain to prevent motor neuron disease operating through a cascade of events as indicated in the schematic, with relevance to amyotrophic lateral sclerosis (ALS). We propose that over-expression of CAST in motor neurons of hSOD1(G93A) mice inhibits activation of CDK5, breakdown of cytoskeletal proteins (NFs, MAP2 and Tau) and regulatory molecules (Cam Kinase IV, Calcineurin A), and disease-causing proteins (TDP-43, α-Synuclein and Huntingtin) to prevent neuronal loss and delay neurological deficits. In our experiments, CAST could also inhibit cleavage of Bid, Bax, AIF to prevent mitochondrial, ER and lysosome-mediated cell death mechanisms. Similarly, CAST over-expression in neurons attenuated pathological effects of TDP-43, α-synuclein and Huntingtin. These results suggest a potential value of specific small molecule inhibitors of calpains in delaying the development of ALS. Read the Editorial Highlight for this article on page 140.

Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits.

Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimer's disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B inhibition on lysosomal proteases suggests that enhancing lysosomal proteolysis improves the overall environment of the lysosome and its clearance functions, which may be possibly relevant to a broader range of lysosomal disorders beyond Alzheimer's disease.

Specific calpain inhibition by calpastatin prevents tauopathy and neurodegeneration and restores normal lifespan in tau P301L mice.

Tau pathogenicity in Alzheimer's disease and other tauopathies is thought to involve the generation of hyperphosphorylated, truncated, and oligomeric tau species with enhanced neurotoxicity, although the generative mechanisms and the implications for disease therapy are not well understood. Here, we report a striking rescue from mutant tau toxicity in the JNPL3 mouse model of tauopathy. We show that pathological activation of calpains gives rise to a range of potentially toxic forms of tau, directly, and by activating cdk5. Calpain overactivation in brains of these mice is accelerated as a result of the marked depletion of the endogenous calpain inhibitor, calpastatin. When levels of this inhibitor are restored in neurons of JNPL3 mice by overexpressing calpastatin, tauopathy is prevented, including calpain-mediated breakdown of cytoskeletal proteins, cdk5 activation, tau hyperphosphorylation, formation of potentially neurotoxic tau fragments by either calpain or caspase-3, and tau oligomerization. Calpastatin overexpression also prevents loss of motor axons, delays disease onset, and extends survival of JNPL3 mice by 3 months to within the range of normal lifespan. Our findings support the therapeutic promise of highly specific calpain inhibition in the treatment of tauopathies and other neurodegenerative states.

Recent trends in diagnosis and control of Marek's disease (MD) in poultry.

Marek's Disease (MD), caused by Marek's Disease Virus (MDV) is a highly contagious oncogenic and neuropathic disease of chickens responsible for great economic losses to the poultry industry all around the world and characterized by development of CD4+T cell lymphomas as well as infiltration of nerves and visceral organs by lymphocytes. MD is one of the most common lymphoproliferative diseases of chickens which cause mononuclear cell infiltration in one or more of the following tissues: peripheral nerves, gonads, lymphoid organs, iris, muscle, skin and other visceral organs resulting into development of tumours in visceral organs, paralysis of legs, wings and neck, grey eye (iris) or irregular pupil, vision impairment, blindness, skin lesions and immunosuppression, all of which can be accompanied by non-specific signs such as anorexia, weight loss and poor performance. Today there are evolving highly pathogenic isolates of MDV around the world capable of overwhelming the protection from currently employed vaccines. Thus MD poses a big challenge to the welfare and wellbeing of the poultry with increased condemnation of carcass, loss of productivity and quality products, leading to huge economic losses. It is also an immunosuppressive disease and causes increased susceptibility to other infections. The present review discusses in brief about the Marek's disease, its etiology, conventional and advance tools and techniques being used for its diagnosis, prevention and control strategies in poultry.

Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis.

The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-ß peptide (Aß) accumulation, extracellular ß-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aß, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aß40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aß clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.

Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits.

Autophagy, a major degradative pathway for proteins and organelles, is essential for survival of mature neurons. Extensive autophagic-lysosomal pathology in Alzheimer's disease brain contributes to Alzheimer's disease pathogenesis, although the underlying mechanisms are not well understood. Here, we identified and characterized marked intraneuronal amyloid-ß peptide/amyloid and lysosomal system pathology in the Alzheimer's disease mouse model TgCRND8 similar to that previously described in Alzheimer's disease brains. We further establish that the basis for these pathologies involves defective proteolytic clearance of neuronal autophagic substrates including amyloid-ß peptide. To establish the pathogenic significance of these abnormalities, we enhanced lysosomal cathepsin activities and rates of autophagic protein turnover in TgCRND8 mice by genetically deleting cystatin B, an endogenous inhibitor of lysosomal cysteine proteases. Cystatin B deletion rescued autophagic-lysosomal pathology, reduced abnormal accumulations of amyloid-ß peptide, ubiquitinated proteins and other autophagic substrates within autolysosomes/lysosomes and reduced intraneuronal amyloid-ß peptide. The amelioration of lysosomal function in TgCRND8 markedly decreased extracellular amyloid deposition and total brain amyloid-ß peptide 40 and 42 levels, and prevented the development of deficits of learning and memory in fear conditioning and olfactory habituation tests. Our findings support the pathogenic significance of autophagic-lysosomal dysfunction in Alzheimer's disease and indicate the potential value of restoring normal autophagy as an innovative therapeutic strategy for Alzheimer's disease.

Induction of autophagy by cystatin C: a mechanism that protects murine primary cortical neurons and neuronal cell lines.

Cystatin C (CysC) expression in the brain is elevated in human patients with epilepsy, in animal models of neurodegenerative conditions, and in response to injury, but whether up-regulated CysC expression is a manifestation of neurodegeneration or a cellular repair response is not understood. This study demonstrates that human CysC is neuroprotective in cultures exposed to cytotoxic challenges, including nutritional-deprivation, colchicine, staurosporine, and oxidative stress. While CysC is a cysteine protease inhibitor, cathepsin B inhibition was not required for the neuroprotective action of CysC. Cells responded to CysC by inducing fully functional autophagy via the mTOR pathway, leading to enhanced proteolytic clearance of autophagy substrates by lysosomes. Neuroprotective effects of CysC were prevented by inhibiting autophagy with beclin 1 siRNA or 3-methyladenine. Our findings show that CysC plays a protective role under conditions of neuronal challenge by inducing autophagy via mTOR inhibition and are consistent with CysC being neuroprotective in neurodegenerative diseases. Thus, modulation of CysC expression has therapeutic implications for stroke, Alzheimer's disease, and other neurodegenerative disorders.

Neuronal apoptosis and autophagy cross talk in aging PS/APP mice, a model of Alzheimer's disease.

Mechanisms of neuronal loss in Alzheimer's disease (AD) are poorly understood. Here we show that apoptosis is a major form of neuronal cell death in PS/APP mice modeling AD-like neurodegeneration. Pyknotic neurons in adult PS/APP mice exhibited apoptotic changes, including DNA fragmentation, caspase-3 activation, and caspase-cleaved alpha-spectrin generation, identical to developmental neuronal apoptosis in wild-type mice. Ultrastructural examination using immunogold cytochemistry confirmed that activated caspase-3-positive neurons also exhibited chromatin margination and condensation, chromatin balls, and nuclear membrane fragmentation. Numbers of apoptotic profiles in both cortex and hippocampus of PS/APP mice compared with age-matched controls were twofold to threefold higher at 6 months of age and eightfold higher at 21 to 26 months of age. Additional neurons undergoing dark cell degeneration exhibited none of these apoptotic features. Activated caspase-3 and caspase-3-cleaved spectrin were abundant in autophagic vacuoles, accumulating in dystrophic neurites of PS/APP mice similar to AD brains. Administration of the cysteine protease inhibitor, leupeptin, promoted accumulation of autophagic vacuoles containing activated caspase-3 in axons of PS/APP mice and, to a lesser extent, in those of wild-type mice, implying that this pro-apoptotic factor is degraded by autophagy. Leupeptin-induced autophagic impairment increased the number of apoptotic neurons in PS/APP mice. Our findings establish apoptosis as a mode of neuronal cell death in aging PS/APP mice and identify the cross talk between autophagy and apoptosis, which influences neuronal survival in AD-related neurodegeneration.

Autophagy induction and autophagosome clearance in neurons: relationship to autophagic pathology in Alzheimer's disease.

Macroautophagy, a major pathway for organelle and protein turnover, has been implicated in the neurodegeneration of Alzheimer's disease (AD). The basis for the profuse accumulation of autophagic vacuoles (AVs) in affected neurons of the AD brain, however, is unknown. In this study, we show that constitutive macroautophagy in primary cortical neurons is highly efficient, because newly formed autophagosomes are rapidly cleared by fusion with lysosomes, accounting for their scarcity in the healthy brain. Even after macroautophagy is strongly induced by suppressing mTOR (mammalian target of rapamycin) kinase activity with rapamycin or nutrient deprivation, active cathepsin-positive autolysosomes rather than LC3-II-positive autophagosomes predominate, implying efficient autophagosome clearance in healthy neurons. In contrast, selectively impeding late steps in macroautophagy by inhibiting cathepsin-mediated proteolysis within autolysosomes with cysteine- and aspartyl-protease inhibitors caused a marked accumulation of electron-dense double-membrane-limited AVs, containing cathepsin D and incompletely degraded LC3-II in perikarya and neurites. Similar structures accumulated in large numbers when fusion of autophagosomes with lysosomes was slowed by disrupting their transport on microtubules with vinblastine. Finally, we find that the autophagic vacuoles accumulating after protease inhibition or prolonged vinblastine treatment strongly resembled AVs that collect in dystrophic neurites in the AD brain and in an AD mouse model. We conclude that macroautophagy is constitutively active and highly efficient in healthy neurons and that the autophagic pathology observed in AD most likely arises from impaired clearance of AVs rather than strong autophagy induction alone. Therapeutic modulation of autophagy in AD may, therefore, require targeting late steps in the autophagic pathway.

Macroautophagy--a novel Beta-amyloid peptide-generating pathway activated in Alzheimer's disease.

Macroautophagy, which is a lysosomal pathway for the turnover of organelles and long-lived proteins, is a key determinant of cell survival and longevity. In this study, we show that neuronal macroautophagy is induced early in Alzheimer's disease (AD) and before beta-amyloid (Abeta) deposits extracellularly in the presenilin (PS) 1/Abeta precursor protein (APP) mouse model of beta-amyloidosis. Subsequently, autophagosomes and late autophagic vacuoles (AVs) accumulate markedly in dystrophic dendrites, implying an impaired maturation of AVs to lysosomes. Immunolabeling identifies AVs in the brain as a major reservoir of intracellular Abeta. Purified AVs contain APP and beta-cleaved APP and are highly enriched in PS1, nicastrin, and PS-dependent gamma-secretase activity. Inducing or inhibiting macroautophagy in neuronal and nonneuronal cells by modulating mammalian target of rapamycin kinase elicits parallel changes in AV proliferation and Abeta production. Our results, therefore, link beta-amyloidogenic and cell survival pathways through macroautophagy, which is activated and is abnormal in AD.

Large cell variant of small cell carcinoma, hypercalcemic type, of primary peritoneal origin.

BACKGROUND: Large cell variant of small cell carcinoma hypercalcemic type (SCC-HT) is extremely rare. All reported cases involved an ovary, and one with primary peritoneal origin has not been described. Also, convincing neuroendocrine granules have not been illustrated. CASE: A 35-year-old woman underwent an exploratory laparotomy for leiomyomas. Intraoperative impression of peritoneal carcinomatosis was confirmed on frozen section. TAH/BSO, debulking/omentectomy followed. The tumor was present on the pelvic/abdominal peritoneum. The normal-sized ovaries were free of tumor grossly. The tumor had features of large cell variant of SCC-HT, described in the ovary. Furthermore, unequivocal neuroendocrine granules were present. The patient received standard chemotherapy for SCC. At 22 months she is NED. CONCLUSION: SCC-HT should be considered in the differential diagnosis of primary neoplasms of the peritoneum.

Antimycobacterial activity of the antiinflammatory agent diclofenac sodium, and its synergism with streptomycin

Diclofenac sódico, um agente antiinflamatório, mostrou ação inibitória marcante contra isolados clínicos de Mycobacterium tuberculosis sensíveis e resistentes à drogas, bem como contra outras micobactérias. A droga foi testada in vitro contra 45 cepas diferentes de micobactérias, sendo que a maioria foi inibida pela droga na concentração de 10-25 µg/ml. Quando testado in vitro, diclofenac injetado em ratos albinos da linhagem Swiss, na proporção de 10 µg/g de peso corporal, provocou proteção significativa dos animais desafiados com metade da dose letal de M. tuberculosis H37 Rv 102. De acordo com o teste c2, os dados in vivo foram altamente significativos (p < 0,01). Diclofenac foi posteriormente testado quanto ao sinergismo com a droga antimicobacteriana convencional estreptomicina, frente a M. smegmatis 798. Quando comparado aos seus efeitos individuais, o sinergismo foi estatisticamente significativo (p < 0,05). Através da análise checkerboard, o índice fracional de concentração inibitória para essa combinação foi 0,37, confirmando o sinergismo.

Binding of cystatin C to Alzheimer's amyloid beta inhibits in vitro amyloid fibril formation.

The colocalization of cystatin C, an inhibitor of cysteine proteases, with amyloid beta (Abeta) in parenchymal and vascular amyloid deposits in brains of Alzheimer's disease (AD) patients may reflect cystatin C involvement in amyloidogenesis. We therefore sought to determine the association of cystatin C with Abeta. Immunofluorescence analysis of transfected cultured cells demonstrated colocalization of cystatin C and beta amyloid precursor protein (betaAPP) intracellularly and on the cell surface. Western blot analysis of immunoprecipitated cell lysate or medium proteins revealed binding of cystatin C to full-length betaAPP and to secreted betaAPP (sbetaAPP). Deletion mutants of betaAPP localized the cystatin C binding site within betaAPP to the Abeta region. Cystatin C association with betaAPP resulted in increased sbetaAPP but did not affect levels of secreted Abeta. Analysis of the association of cystatin C and Abeta demonstrated a specific, saturable and high affinity binding between cystatin C and both Abeta(1-42) and Abeta(1-40). Notably, cystatin C association with Abeta results in a concentration-dependent inhibition of Abeta fibril formation.

Parachordoma or chordoma periphericum? Case report of a tumor of the thoracic wall.

We report the findings from an aspiration biopsy and resection of a chordoma-like tumorous mass in the wall of the thorax of a 36-yr-old man with immunohistochemical, ultrastructural, and cytogenetic studies. The 4-cm oval tumor was an incidental finding on physical examination, and no other lesions were identified after comprehensive radiologic studies. The aspirate was composed of sheets and nests of cells with distinct borders in a myxoid and fibrillary extracellular matrix. The neoplastic cells were uniform and round or polygonal with abundant pale blue vacuolated cytoplasm and small round, central or eccentric nuclei. On electron microscopy, mitochondrial rough endoplasmic reticulum complexes were seen in neoplastic cells. These features were similar to those of a conventional chordoma. However, the cytogenetic pattern, 43, XY ,-1, -2, der (5)t(1p;5q), -6, add(8p) ,add(10q), was not typical. In addition, the neoplastic cells were positive for vimentin, S-100, AE1/AE3, CAM 5.2, and CK 19; were focally positive for EMA and smooth muscle actin; and were negative for cytokeratin 1 and 10 (34 beta E12), CK 7, CK 8 (35H 11B), CK 17, and CK 20. The cytogenetic and immunohistochemical patterns were different from conventional chordoma and its peripheral counterpart, chordoma periphericum, suggesting the diagnosis of parachordoma. To the best of our knowledge, this is the first report of fine-needle aspiration of this newly defined and rare entity.

Size fractions of ambient particulate matter induce granulocyte macrophage colony-stimulating factor in human bronchial epithelial cells by mitogen-activated protein kinase pathways.

Environmental pollutants, including ambient particulate matter (PM), increase respiratory morbidity. Studies of model PM particles, including residual oil fly ash and freshly generated diesel exhaust particles, have demonstrated that PM affects inflammatory airway responses. Neither of these particles completely represents ambient PM, and therefore questions remain about ambient particulates. We hypothesized that ambient PM of different size fractions collected from an urban environment (New York City air), would activate primary culture human bronchial epithelial cells (HBECs). Because of the importance of granulocyte-macrophage colony-stimulating factor (GM-CSF) on inflammatory and immunomodulatory processes, we focused our studies on this cytokine. We demonstrated that the smallest size fraction (ultrafine/fine; < 0.18 micro m) of ambient PM (11 micro g/cm(2)), upregulated GM-CSF production (2-fold increase). The absence of effect of carbon particles of similar size, and the day-to-day variation in response, suggested that the chemical composition, but not the particle itself, was necessary for GM-CSF induction. Activation of the extracellular signal-regulated kinase and the p38 mitogen-activated protein kinase was associated with, and necessary for, GM-CSF release. These studies serve to corroborate and extend those on model particles. Moreover, they emphasize the role of the smallest size ambient particles in airway epithelial cell responses.

Tumoral non-amyloidotic monoclonal immunoglobulin light chain deposits ('aggregoma'): presenting feature of B-cell dyscrasia in three cases with immunohistochemical and biochemical analyses.

Tumoral monoclonal immunoglobulin (Ig) light chain non-fibrillar deposits ('aggregomas'), which can be considered analogous to solitary light chain amyloidomas, are a rare presenting feature of B-cell dyscrasias. It is not certain if they are truly localized or if in reality they represent an initial expression of a silent systemic non-amyloid light chain deposition disease (LCDD). This report describes three patients, two of whom presented with cervical masses and the third with a solitary lung nodule, each comprising granular aggregates of monoclonal kappa light chain. Extracted deposits from the lymph node of one patient were shown by N-terminal amino acid sequence analysis to belong to the variable-region kappa I (Vkappa I) light chain subgroup, the first reported kappa-LCDD protein encoded by the L9 gene and the first report of an expressed protein related to this gene. Extracted deposits from the lung nodule of the second patient belonged to the Vkappa IV light chain subgroup encoded by the B3 germ line gene. The N-terminal amino acid sequences of the light chains from the aggregomas were compared with the related germ line sequences and to the N-terminal amino acid sequences of the nine other known kappa-LCDD light chains reported thus far from patients with systemic LCDD.