Vasculitides associated with IgG antineutrophil cytoplasmic autoantibodies in childhood

Immunoglobulin (Ig)G antineutrophil cytoplasmic autoantibodies are causally associated with necrotizing vasculitides that are characterized immunopathologically by little or no deposition of immunoreactants, such as Wegener granulomatosis, microscopic polyangiitis, Churg-Strauss angiitis, "renal-limited" vasculitis and a number of drug-induced vasculitides. Clinical routine testing targets the antigens myeloperoxidase and proteinase 3. However, in all of the conditions mentioned, the renal histopathologic findings are indistinguishable. Churg-Strauss angiitis (characterized by necrotizing vasculitis, granulomatous inflammation and tissue eosinophilia), Wegener granulomatosis (characterized by necrotizing vasculitis and granulomatous inflammation) and microscopic polyangiitis (characterized by necrotizing vasculitis) often present with fever, weight loss and a multisystem involvement (ear, nose, throat, lung, eyes, peripheral nerve and heart). Fifty years ago these conditions were very often fatal within 6 months of diagnosis. The introduction of corticosteroids and cyclophosphamide has resulted in a dramatic clinical benefit. Patients who develop treatment-related morbidity can be switched from cyclophosphamide to azathioprine after achieving remission. In patients with less severe disease, methotrexate achieves remission with a success rate similar to that of cyclophosphamide. Plasma exchange, in association with immunosuppression, is likely to be a beneficial therapy for patients with severe kidney disease or pulmonary hemorrhage.

High-level cytoplasmic cyclin D1 expression in lymph node metastases from prostate cancer independently predicts early biochemical failure and death in surgically treated patients

To test the prognostic significance of cyclin D1 in nodal-positive prostate cancer.

Alba-domain proteins of Trypanosoma brucei are cytoplasmic RNA-binding proteins that interact with the translation machinery

Trypanosoma brucei and related pathogens transcribe most genes as polycistronic arrays that are subsequently processed into monocistronic mRNAs. Expression is frequently regulated post-transcriptionally by cis-acting elements in the untranslated regions (UTRs). GPEET and EP procyclins are the major surface proteins of procyclic (insect midgut) forms of T. brucei. Three regulatory elements common to the 3' UTRs of both mRNAs regulate mRNA turnover and translation. The glycerol-responsive element (GRE) is unique to the GPEET 3' UTR and regulates its expression independently from EP. A synthetic RNA encompassing the GRE showed robust sequence-specific interactions with cytoplasmic proteins in electromobility shift assays. This, combined with column chromatography, led to the identification of 3 Alba-domain proteins. RNAi against Alba3 caused a growth phenotype and reduced the levels of Alba1 and Alba2 proteins, indicative of interactions between family members. Tandem-affinity purification and co-immunoprecipitation verified these interactions and also identified Alba4 in sub-stoichiometric amounts. Alba proteins are cytoplasmic and are recruited to starvation granules together with poly(A) RNA. Concomitant depletion of all four Alba proteins by RNAi specifically reduced translation of a reporter transcript flanked by the GPEET 3' UTR. Pulldown of tagged Alba proteins confirmed interactions with poly(A) binding proteins, ribosomal protein P0 and, in the case of Alba3, the cap-binding protein eIF4E4. In addition, Alba2 and Alba3 partially cosediment with polyribosomes in sucrose gradients. Alba-domain proteins seem to have exhibited great functional plasticity in the course of evolution. First identified as DNA-binding proteins in Archaea, then in association with nuclear RNase MRP/P in yeast and mammalian cells, they were recently described as components of a translationally silent complex containing stage-regulated mRNAs in Plasmodium. Our results are also consistent with stage-specific regulation of translation in trypanosomes, but most likely in the context of initiation.

Diagnostic value of biopsies in identifying cytoplasmic antineutrophil cytoplasmic antibody-negative localized Wegener's granulomatosis presenting primarily with sinonasal disease

A substantial proportion of Wegener's disease (WG) patients present with localized disease of the upper airways, i.e., sinonasal and other ear/nose/throat (ENT) symptoms. Because of the oligosymptomatic presentation a timely diagnosis of this potentially fatal disease is challenging. This study evaluates diagnostic peculiarities between WG in its localized and generalized form of the disease.

Perinuclear antineutrophilic cytoplasmic antibody and response to treatment in diarrheic dogs with food responsive disease or inflammatory bowel disease

The goal of this study was to investigate the correlation between perinuclear antineutrophilic cytoplasmic antibody (pANCA) and clinical scores before and after treatment in diarrheic dogs with food-responsive disease (FRD) or inflammatory bowel disease (IBD). pANCA serology was evaluated prospectively by indirect immunofluorescence in 65 dogs with signs of gastrointestinal disease, and if positive, pANCA antibody titers were determined. Thirty-nine dogs with FRD responded to a novel diet, and 26 dogs with IBD were treated with corticosteroids. The severity of clinical signs was scored by means of a canine IBD activity index (CIBDAI). At initial examination, a significantly (P = .002) higher percentage of dogs were pANCA-positive in the FRD group (62%) compared with the IBD group (23%). pANCA titers were significantly higher (P = .003) before treatment in the FRD group (median titer 100) compared with the IBD group (median titer 1). However, there was no difference in pANCA titers between the groups after respective treatments because dogs in the IBD group had a significant increase in pANCA titer after treatment. The CIBDAI score decreased significantly (P < .001) after treatment in both groups (74% moderate to severe in FRD dogs before versus 8% after treatment; 85% moderate to severe in IBD dogs before versus 32% after treatment). There was no correlation between pANCA status in FRD or IBD dogs before treatment and scores for CIBDAI, endoscopy, or histopathology before or after treatment, except for the endoscopic duodenal score in dogs with FRD after treatment (P = .03). A positive pANCA test before therapy may aid in the diagnosis of FRD.

The cytoplasmic domain of the ligand ephrinB2 is required for vascular morphogenesis but not cranial neural crest migration

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of vascular morphogenesis. EphrinB2 may have an active signaling role, resulting in bi-directional signal transduction downstream of both ephrinB2 and Eph receptors. To separate the ligand and receptor-like functions of ephrinB2 in mice, we replaced the endogenous gene by cDNAs encoding either carboxyterminally truncated (ephrinB2(DeltaC)) or, as a control, full-length ligand (ephrinB2(WT)). While homozygous ephrinB2(WT/WT) animals were viable and fertile, loss of the ephrinB2 cytoplasmic domain resulted in midgestation lethality similar to ephrinB2 null mutants (ephrinB2(KO)). The truncated ligand was sufficient to restore guidance of migrating cranial neural crest cells, but ephrinB2(DeltaC/DeltaC) embryos showed defects in vasculogenesis and angiogenesis very similar to those observed in ephrinB2(KO/KO) animals. Our results indicate distinct requirements of functions mediated by the ephrinB carboxyterminus for developmental processes in the vertebrate embryo.

Hypoxia increases cytoplasmic expression of NDRG1, but is insufficient for its membrane localization in human hepatocellular carcinoma

NDRG1 is a hypoxia-inducible protein, whose modulated expression is associated with the progression of human cancers. Here, we reveal that NDRG1 is markedly upregulated in the cytoplasm and on the membrane in human hepatocellular carcinoma (HCC). We demonstrate further that hypoxic stress increases the cytoplasmic expression of NDRG1 in vitro, but does not result in its localization on the plasma membrane. However, grown within an HCC-xenograft in vivo, cells express NDRG1 in the cytoplasm and on the plasma membrane. In conclusion, hypoxia is a potent inducer of NDRG1 in HCCs, albeit requiring additional stimuli within the tumour microenvironment for its recruitment to the membrane.

Point mutations in the stem region and the fourth AAA domain of cytoplasmic dynein heavy chain partially suppress the phenotype of NUDF/LIS1 loss in Aspergillus nidulans

Cytoplasmic dynein performs multiple cellular tasks but its regulation remains unclear. The dynein heavy chain has a N-terminal stem that binds to other subunits and a C-terminal motor unit that contains six AAA (ATPase associated with cellular activities) domains and a microtubule-binding site located between AAA4 and AAA5. In Aspergillus nidulans, NUDF (a LIS1 homolog) functions in the dynein pathway, and two nudF6 partial suppressors were mapped to the nudA dynein heavy chain locus. Here we identified these two mutations. The nudAL1098F mutation resides in the stem region, and nudAR3086C is in the end of AAA4. These mutations partially suppress the phenotype of nudF deletion but do not suppress the phenotype exhibited by mutants of dynein intermediate chain and Arp1. Surprisingly, the stronger DeltanudF suppressor, nudAR3086C, causes an obvious decrease in the basal level of dynein's ATPase activity and an increase in dynein's distribution along microtubules. Thus, suppression of the DeltanudF phenotype may result from mechanisms other than simply the enhancement of dynein's ATPase activity. The fact that a mutation in the end of AAA4 negatively regulates dynein's ATPase activity but partially compensates for NUDF loss indicates the importance of the AAA4 domain in dynein regulation in vivo.

Phosphorylation of serine 4642 in the C-terminus of plectin by MNK2 and PKA modulates its interaction with intermediate filaments

Plectin is a versatile cytolinker of the plakin family conferring cell resilience to mechanical stress in stratified epithelia and muscles. It acts as a critical organizer of the cytoskeletal system by tethering various intermediate filament (IF) networks through its C-terminal IF-binding domain (IFBD). Mutations affecting the IFBD cause devastating human diseases. Here, we show that serine 4642, which is located in the extreme C-terminus of plectin, is phosphorylated in different cell lines. Phosphorylation of S4642 decreased the ability of plectin IFBD to associate with various IFs, as assessed by immunofluorescence microscopy and cell fractionation studies, as well as in yeast two-hybrid assays. Plectin phosphorylated at S4642 was reduced at sites of IF network anchorage along cell-substrate contacts in both skin and cultured keratinocytes. Treatment of SK-MEL-2 and HeLa cells with okadaic acid increased plectin S4642 phosphorylation, suggesting that protein phosphatase 2A dephosphorylates this residue. Moreover, plectin S4642 phosphorylation was enhanced after cell treatment with EGF, phorbol ester, sorbitol and 8-bromo-cyclic AMP, as well as during wound healing and protease-mediated cell detachment. Using selective protein kinase inhibitors, we identified two different kinases that modulate the phosphorylation of plectin S4642 in HeLa cells: MNK2, which is downstream of the ERK1/2-dependent MAPK cascade, and PKA. Our study indicates that phosphorylation of S4642 has an important regulatory role in the interaction of plectin with IFs and identifies a novel link between MNK2 and the cytoskeleton.

The growing galectin network in colon cancer and clinical relevance of cytoplasmic galectin-3 reactivity

BACKGROUND/AIM Human lectins translate sugar-encoded signals of cell surface glycoconjugates into biological effects, and this is what is known for the adhesion/growth-regulatory galectins. In addition, the multifunctional members of this group can be intracellular, binding to distinct proteins. The presence of galectins and galectin reactivity were exemplarily studied in the present article. MATERIALS AND METHODS We combined immuno- and lectin histochemical monitoring in colon cancer on tissue arrays. RESULTS Intracellular presence of galectins-7 and -9 in colon cancer is detected, extending the previously known set of five expressed lectins this tumor type. The assumed significance of intracellular galectin presence, e.g. for an interplay with BCL2, β-catenin, oncogenic KRAS or synexin, is underscored by respective staining with labeled galectin-3. Statistical significance was obtained for galectin-3 staining with respect to tumor differentiation (p=0.0376), lymph node metastasis (p=0.0069) and lymphatic invasion (p=0.0156). Survival was correlated to staining, galectin-3 reactivity indicating a favorable prognosis (p=0.0183), albeit not as an independent marker. No correlation to KRAS/BRAF status was detected. CONCLUSION These results encourage further testing of labeled human galectins as probes and immunohistochemical fingerprinting instead of measuring single or few activities, in colon cancer and other tumor types.

Pneumocystis carinii pneumonia during immunosuppressive therapy for antineutrophil cytoplasmic autoantibody-positive vasculitis.

We describe two human immunodeficiency virus-negative patients who developed Pneumocystis carinii pneumonia (PCP) during immunosuppressive therapy for antineutrophil cytoplasmic autoantibody-positive vasculitis and review the literature regarding the pathogenesis and frequency of PCP. The recent application of DNA amplification techniques suggests that PCP developing in immunocompromised individuals does not necessarily result from reactivation of a dormant focus, but may arise as de novo infection after exposure to an exogenous source of P carinii. In addition, several reports about clusters of PCP cases raise concern about the risk of a nosocomial transmission of P carinii. Therefore, PCP should be added to the list of bronchopulmonary complications in patients with antineutrophil cytoplasmic autoantibody-positive vasculitis who are receiving long-term steroid therapy.

Late Quaternary history of North Eurasian Norway spruce ( Picea abies ) and Siberian spruce ( Picea obovata ) inferred from macrofossils, pollen and cytoplasmic DNA variation

Aim We used combined palaeobotanical and genetic data to assess whether Norway spruce (Picea abies) and Siberian spruce (Picea obovata), two major components of the Eurasian boreal forests, occupied separate glacial refugia, and to test previous hypotheses on their distinction, geographical delimitation and introgression. Location The range of Norway spruce in northern Europe and Siberian spruce in northern Asia. Methods Pollen data and recently compiled macrofossil records were summarized for the Last Glacial Maximum (LGM), late glacial and Holocene. Genetic variation was assessed in 50 populations using one maternally (mitochondrial nad1) and one paternally (chloroplast trnT–trnL) inherited marker and analysed using spatial analyses of molecular variance (SAMOVA). Results Macrofossils showed that spruce was present in both northern Europe and Siberia at the LGM. Congruent macrofossil and pollen data from the late glacial suggested widespread expansions of spruce in the East European Plain, West Siberian Plain, southern Siberian mountains and the Baikal region. Colonization was largely completed during the early Holocene, except in the formerly glaciated area of northern Europe. Both DNA markers distinguished two highly differentiated groups that correspond to Norway spruce and Siberian spruce and coincide spatially with separate LGM spruce occurrences. The division of the mtDNA variation was geographically well defined and occurred to the east of the Ural Mountains along the Ob River, whereas the cpDNA variation showed widespread admixture. Genetic diversity of both DNA markers was higher in western than in eastern populations. Main conclusions North Eurasian Norway spruce and Siberian spruce are genetically distinct and occupied separate LGM refugia, Norway spruce on the East European Plain and Siberian spruce in southern Siberia, where they were already widespread during the late glacial. They came into contact in the basin of the Ob River and probably hybridized. The lower genetic diversity in the eastern populations may indicate that Siberian spruce suffered more from past climatic fluctuations than Norway spruce.