Differential distribution of MAP1a and aldolase c in adult mouse cerebellum.

MAP1a is a microtubule-associated protein with an apparent molecular weight of 360 kDa that is found in the axonal and dendritic processes of neurons. Two monoclonal anti-MAP1a antibodies anti-A and anti-BW6, revealed different epitope distributions in the adult mouse cerebellum. Anti-A stained Purkinje and granule cells uniformly throughout the cerebellum. In contrast, anti-BW6 selectively stained the dendriites of a subset of Purkinje cells, revealing parasagittal bands of immunoreactivity in the molecular layer. The compartmentation of the BW6 epitope was compared to the Purkine cells as revealed by immunostaining with anti-zebrin II, a well known antigen expressed selectively by bands of Purkinje cells. The anti-BW6 staining pattern was complementary to the zebrin II bands, the zebrin II- Purkinjke cells having BW6+ dendrites. These results demonstrate that MAP1a is present in two forms in the mouse cerebellum, one of which is segregated into parasagittal bands. This may indicate a unique MAP1a isoform or may reflect differences in the metabolic states of Purkinje cell classes, and regional differences in their functions.

Beta-catenin is dispensable for hematopoiesis and lymphopoiesis.

Beta-catenin-mediated Wnt signaling has been suggested to be critically involved in hematopoietic stem cell maintenance and development of T and B cells in the immune system. Unexpectedly, here we report that inducible Cre-loxP-mediated inactivation of the beta-catenin gene in bone marrow progenitors does not impair their ability to self-renew and reconstitute all hematopoietic lineages (myeloid, erythroid, and lymphoid), even in competitive mixed chimeras. In addition, both thymocyte survival and antigen-induced proliferation of peripheral T cells is beta-catenin independent. In contrast to earlier reports, these data exclude an essential role for beta-catenin during hematopoiesis and lymphopoiesis.

Dynamic regulation of notch 1 and notch 2 surface expression during T cell development and activation revealed by novel monoclonal antibodies.

It is well established that Notch signaling plays a critical role at multiple stages of T cell development and activation. However, detailed analysis of the cellular and molecular events associated with Notch signaling in T cells is hampered by the lack of reagents that can unambiguously measure cell surface Notch receptor expression. Using novel rat mAbs directed against the extracellular domains of Notch1 and Notch2, we find that Notch1 is already highly expressed on common lymphoid precursors in the bone marrow and remains at high levels during intrathymic maturation of CD4(-)CD8(-) thymocytes. Notch1 is progressively down-regulated at the CD4(+)CD8(+) and mature CD4(+) or CD8(+) thymic stages and is expressed at low levels on peripheral T cells. Immunofluorescence staining of thymus cryosections further revealed a localization of Notch1(+)CD25(-) cells adjacent to the thymus capsule. Notch1 was up-regulated on peripheral T cells following activation in vitro with anti-CD3 mAbs or infection in vivo with lymphocytic chorio-meningitis virus or Leishmania major. In contrast to Notch1, Notch2 was expressed at intermediate levels on common lymphoid precursors and CD117(+) early intrathymic subsets, but disappeared completely at subsequent stages of T cell development. However, transient up-regulation of Notch2 was also observed on peripheral T cells following anti-CD3 stimulation. Collectively our novel mAbs reveal a dynamic regulation of Notch1 and Notch2 surface expression during T cell development and activation. Furthermore they provide an important resource for future analysis of Notch receptors in various tissues including the hematopoietic system.

The human Ia-associated invariant chain is synthesized in Ia-negative B cell variants and is not expressed on the cell surface of both Ia-negative and Ia-positive parental cells.

The expression of Ia-associated human Invariant (In) chain glycoproteins was studied in the Raji B cells as well as in their RJ 2.2.5 Ia-negative derived variant cells by using a specific rabbit anti-human In chain antiserum. Two-dimensional gel electrophoresis of immunoprecipitates from either biosynthetically labeled or surface labeled cells were analyzed. In addition, flow microfluorometric analysis of stained cells was performed. The results indicate that the In chain is constitutively produced in the Ia-negative B cell variant. Moreover, it appears that several forms of In chain-related molecules, with different charges and distinct m.w. are equally expressed in Ia-positive and Ia-negative B cells. Finally, no evidence could be obtained that the In molecular family was expressed on the cell surface of Ia-positive Raji and Ia-negative RJ 2.2.5 cells.

Repression of flagellar genes in exponential phase by CsgD and CpxR, two crucial modulators of Escherichia coli biofilm formation.

Escherichia coli adapts its lifestyle to the variations of environmental growth conditions, swapping between swimming motility or biofilm formation. The stationary-phase sigma factor RpoS is an important regulator of this switch, since it stimulates adhesion and represses flagellar biosynthesis. By measuring the dynamics of gene expression, we show that RpoS inhibits the transcription of the flagellar sigma factor, FliA, in exponential growth phase. RpoS also partially controls the expression of CsgD and CpxR, two transcription factors important for bacterial adhesion. We demonstrate that these two regulators repress the transcription of fliA, flgM, and tar and that this regulation is dependent on the growth medium. CsgD binds to the flgM and fliA promoters around their -10 promoter element, strongly suggesting direct repression. We show that CsgD and CpxR also affect the expression of other known modulators of cell motility. We propose an updated structure of the regulatory network controlling the choice between adhesion and motility.

Effect of oxidized cholesterol on age-associated changes to immune parameters in spleen lymphocytes and peritoneal exudate cells derived from rats

The effects of oxidized cholesterol on immune parameters were examined by using spleen lymphocytes and peritoneal exudate cells (PEC) derived from 5-week- (Young) and 9-month-old (Adult) rats. The immunoglobulin (Ig) G and IgM production was inhibited by oxidized cholesterol in the rats of both ages when lymphocytes were exposed to 30 micrograms/ml of oxidized cholesterol for 24 hr. The intracellular IgA level was also lowered by 30 micrograms/ml of oxidized cholesterol, irrespective of age. In contrast, IgE production was significantly increased by the addition of 30 micrograms/ml of oxidized cholesterol in only young lymphocytes. Moreover, oxidized cholesterol enhanced the intracellular histamine accumulation in only adult PEC, although the total histamine level produced by PEC was similar in the rats of both ages. These results thus suggest the possibility that oxidized cholesterol can have different effects on the age-related modulation of immune functions such as Igs production and histamine release.

Simultaneous loss of beta- and gamma-catenin does not perturb hematopoiesis or lymphopoiesis.

Hematopietic stem cells (HSCs) maintain life-long hematopoiesis in the bone marrow via their ability to self-renew and to differentiate into all blood lineages. Although a central role for the canonical wnt signaling pathway has been suggested in HSC self-renewal as well as in the development of B and T cells, conditional deletion of beta-catenin (which is considered to be essential for Wnt signaling) has no effect on hematopoiesis or lymphopoiesis. Here, we address whether this discrepancy can be explained by a redundant and compensatory function of gamma-catenin, a close homolog of beta-catenin. Unexpectedly, we find that combined deficiency of beta- and gamma-catenin in hematopoietic progenitors does not impair their ability to self-renew and to reconstitute all myeloid, erythroid, and lymphoid lineages, even in competitive mixed chimeras and serial transplantations. These results exclude an essential role for canonical Wnt signaling (as mediated by beta- and/or gamma-catenin) during hematopoiesis and lymphopoiesis.

Is there an advantage to be gained from adding digital image cytometry of brush cytology to a standard biopsy protocol in patients with Barrett's esophagus?

BACKGROUND AND STUDY AIMS: The current gold standard in Barrett's esophagus monitoring consists of four-quadrant biopsies every 1-2 cm in accordance with the Seattle protocol. Adding brush cytology processed by digital image cytometry (DICM) may further increase the detection of patients with Barrett's esophagus who are at risk of neoplasia. The aim of the present study was to assess the additional diagnostic value and accuracy of DICM when added to the standard histological analysis in a cross-sectional multicenter study of patients with Barrett's esophagus in Switzerland. METHODS: One hundred sixty-four patients with Barrett's esophagus underwent 239 endoscopies with biopsy and brush cytology. DICM was carried out on 239 cytology specimens. Measures of the test accuracy of DICM (relative risk, sensitivity, specificity, likelihood ratios) were obtained by dichotomizing the histopathology results (high-grade dysplasia or adenocarcinoma vs. all others) and DICM results (aneuploidy/intermediate pattern vs. diploidy). RESULTS: DICM revealed diploidy in 83% of 239 endoscopies, an intermediate pattern in 8.8%, and aneuploidy in 8.4%. An intermediate DICM result carried a relative risk (RR) of 12 and aneuploidy a RR of 27 for high-grade dysplasia/adenocarcinoma. Adding DICM to the standard biopsy protocol, a pathological cytometry result (aneuploid or intermediate) was found in 25 of 239 endoscopies (11%; 18 patients) with low-risk histology (no high-grade dysplasia or adenocarcinoma). During follow-up of 14 of these 18 patients, histological deterioration was seen in 3 (21%). CONCLUSION: DICM from brush cytology may add important information to a standard biopsy protocol by identifying a subgroup of BE-patients with high-risk cellular abnormalities.

Tumor necrosis factor and transforming growth factor β regulate clock genes by controlling the expression of the cold inducible RNA-binding protein (CIRBP).

The circadian clock drives the rhythmic expression of a broad array of genes that orchestrate metabolism, sleep wake behavior, and the immune response. Clock genes are transcriptional regulators engaged in the generation of circadian rhythms. The cold inducible RNA-binding protein (CIRBP) guarantees high amplitude expression of clock. The cytokines TNF and TGFβ impair the expression of clock genes, namely the period genes and the proline- and acidic amino acid-rich basic leucine zipper (PAR-bZip) clock-controlled genes. Here, we show that TNF and TGFβ impair the expression of Cirbp in fibroblasts and neuronal cells. IL-1β, IL-6, IFNα, and IFNγ do not exert such effects. Depletion of Cirbp is found to increase the susceptibility of cells to the TNF-mediated inhibition of high amplitude expression of clock genes and modulates the TNF-induced cytokine response. Our findings reveal a new mechanism of cytokine-regulated expression of clock genes.

IRF6 is a mediator of Notch pro-differentiation and tumour suppressive function in keratinocytes.

While the pro-differentiation and tumour suppressive functions of Notch signalling in keratinocytes are well established, the underlying mechanisms remain poorly understood. We report here that interferon regulatory factor 6 (IRF6), an IRF family member with an essential role in epidermal development, is induced in differentiation through a Notch-dependent mechanism and is a primary Notch target in keratinocytes and keratinocyte-derived SCC cells. Increased IRF6 expression contributes to the impact of Notch activation on growth/differentiation-related genes, while it is not required for induction of 'canonical' Notch targets like p21(WAF1/Cip1), Hes1 and Hey1. Down-modulation of IRF6 counteracts differentiation of primary human keratinocytes in vitro and in vivo, promoting ras-induced tumour formation. The clinical relevance of these findings is illustrated by the strikingly opposite pattern of expression of Notch1 and IRF6 versus epidermal growth factor receptor in a cohort of clinical SCCs, as a function of their grade of differentiation. Thus, IRF6 is a primary Notch target in keratinocytes, which contributes to the role of this pathway in differentiation and tumour suppression.

Intrauterine growth restriction and absent or reverse end-diastolic blood flow in umbilical artery (Doppler class II or III): A retrospective study of short- and long-term fetal morbidity and mortality.

OBJECTIVE: Absent or reverse end-diastolic flow (Doppler II/III) in umbilical artery is correlated with poor perinatal outcome, particularly in intrauterine growth restricted (IUGR) fetuses. The optimal timing of delivery is still controversial. We studied the short- and long-term morbidity and mortality among these children associated with our defined management. STUDY DESIGN: Sixty-nine IUGR fetuses with umbilical Doppler II/III were divided into three groups; Group 1, severe early IUGR, no therapeutic intervention (n = 7); Group 2, fetuses with pathological biophysical profile, immediate delivery (n = 35); Group 3, fetuses for which expectant management had been decided (n = 27). RESULTS: In Group 1, stillbirth was observed after a mean delay of 6.3 days. Group 2 delivered at an average of 31.6 weeks and two died in the neonatal period (6%). In Group 3 after a mean delay of 8 days, average gestational age at delivery was 31.7 weeks; two intra uterine and four perinatal deaths were observed (22%). Long-term follow-up revealed no sequelae in 25/31 (81%) and 15/18 (83%), and major handicap occurred in 1 (3%) and 2 patients (11%), respectively, for Groups 2 and 3. CONCLUSION: Fetal mortality was observed in 22% of this high risk group. After a mean period of follow-up of 5 years, 82% of infants showed no sequelae. According to our management, IUGR associated with umbilical Doppler II or III does not show any benefit from an expectant management in term of long-term morbidity.

Induction of transendothelial migration in normal and malignant human T lymphocytes.

Activated CD 3+ enriched human peripheral blood T cells exhibited potent capacity for transendothelial migration through HUVEC layers in the absence of T cell ***. In contrast, malignant human T cell lines *** no or negligible ability of transendothelial migration in the absence of chemoattractants. Time lapse studies of transendothelial migration of activated CD 3+ enriched peripheral blood T cells through a HUVEC layer showed that the first T cells were detected in the lower compartment of a tissue culture insert after 1 hour and that migration increased to reach a maximum of 25 x 10(4) T cells/hr after 24 hours. Adhesion assays of human T cell lines demonstrated that all T cell lines were capable of adhesion to HUVEC and that adhesion of T cells to HUVECs was primarily mediated by CD11a/CD18 and ICAM-1 interactions. Furthermore, transendothelial migration of CD 3+ enriched human peripheral blood T cells was inhibited by pretreating the T cells with anti-CD 18 monoclonal antibodies. The inability of malignant T cells to migrate through HUVEC layers in the absence of chemoattractants was not due to poor motility per se, since both normal and malignant T cells migrated well on extracellular matrix components as determined by using Boyden chambers. Crosslinking of alpha 1 beta 2 and alpha 4 beta 1 with immobilized monoclonal antibodies induced motile behaviour in activated CD 3 enriched human peripheral blood T cells but not in malignant T cell lines. In conclusion, the differences in the ability of transendothelial migration between normal and malignant human T cells in the absence of chemoattractants is primarily due to the differences in the capacity of alpha 1 beta 2 and alpha 4 beta 1 to trigger motile behaviour in the separate cell types.

APRIL is critical for plasmablast survival in the bone marrow and poorly expressed by early-life bone marrow stromal cells.

The persistence of serum IgG antibodies elicited in human infants is much shorter than when such responses are elicited later in life. The reasons for this rapid waning of antigen-specific antibodies elicited in infancy are yet unknown. We have recently shown that adoptively transferred tetanus toxoid (TT)-specific plasmablasts (PBs) efficiently reach the bone marrow (BM) of infant mice. However, TT-specific PBs fail to persist in the early-life BM, suggesting that they fail to receive the molecular signals that support their survival/differentiation. Using a proliferation-inducing ligand (APRIL)- and B-cell activating factor (BAFF) B-lymphocyte stimulator (BLyS)-deficient mice, we demonstrate here that APRIL is a critical factor for the establishment of the adult BM reservoir of anti-TT IgG-secreting cells. Through in vitro analyses of PB/plasma cell (PC) survival/differentiation, we show that APRIL induces the expression of Bcl-X(L) by a preferential binding to heparan sulfate proteoglycans at the surface of CD138(+) cells. Last, we identify BM-resident macrophages as the main cells that provide survival signals to PBs and show that this function is slowly acquired in early life, in parallel to a progressive acquisition of APRIL expression. Altogether, this identifies APRIL as a critical signal for PB survival that is poorly expressed in the early-life BM compartment.

Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.

Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.

Epidermal growth factor and bovine growth hormone stimulate differentiation and myelination of brain cell aggregates in culture.

Bovine growth hormone (bGH) and epidermal growth factor (EGF) increased the activity of ornithine decarboxylase (ODC) in brain cell aggregates cultured in a serum-free chemically defined medium. ODC is considered as a marker of cell growth and differentiation. The effect of bGH and EGF on myelination was investigated by measuring two myelin markers, 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) and myelin basic protein (MBP). EGF treatment at days 2 and 5 caused a dose-dependent increase of both myelin markers at culture day 12. This increase could still be observed at culture day 19, indicating a prolonged action of EGF. The continual presence of bGH in the culture medium produced a large accumulation of MBP at day 19. This effect was dose-dependent and required the presence of triiodothyronine (T3). In contrast, the effect of bGH on CNP activity did not require the presence of T3. This is the first report showing a direct effect of bGH on CNS myelination in vitro and of EGF on both MBP accumulation and ODC activity.