CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases

OBJECTIVE: Dendritic cells (DC) are the only antigen-presenting cells that can activate naive T lymphocytes and initiate a primary immune response. They are also thought to have a role in immune tolerance. DC traffic from the blood to peripheral tissue where they become activated. They then present antigen and the costimulating signals necessary to initiate an immune response. In this study, we investigated the number, subsets, and activation pattern of circulating and intestinal DC from patients with clinically mild ulcerative colitis (UC) or Crohn's disease. METHODS: Patients were recruited, if they were not taking immunosuppressive therapy, and were assessed for clinical severity of their disease using for UC, the Clinical Activity Index, and for Crohn's disease, the Crohn's Disease Activity Index. Blood CD11c(+) and CD11c(-) DC subsets, expression of costimulatory antigens, CD86 and CD40, and the early differentiation/activation antigen, CMRF44, were enumerated by multicolor flow cytometry of lineage negative (lin(-) = CD3(-), CD19(-), CD14(-), CD16(-)) HLA-DR+ DC. These data were compared with age-matched healthy and the disease control groups of chronic noninflammatory GI diseases (cGI), acute noninflammatory GI diseases (aGI), and chronic non-GI inflammation (non-GI). In addition, cryostat sections of colonoscopic biopsies from healthy control patients and inflamed versus noninflamed gut mucosa of inflammatory bowel disease (IBD) patients were examined for CD86(+) and CD40(+)lin(-) cells. RESULTS: Twenty-one Crohn's disease and 25 UC patients, with mean Crohn's Disease Activity Index of 98 and Clinical Activity Index of 3.1, and 56 healthy controls, five cGI, five aGI, and six non-GI were studied. CD11c(+) and CD11c(-) DC subsets did not differ significantly between Crohn's, UC, and healthy control groups. Expression of CD86 and CD40 on freshly isolated blood DC from Crohn's patients appeared higher (16.6%, 31%) and was significantly higher in UC (26.6%, 46.3%) versus healthy controls (5.5%, 25%) (p = 0.004, p = 0.012) and non-GI controls (10.2%, 22.8%) (p = 0.012, p = 0.008), but not versus cGI or aGI controls. CD86(+) and CD40(+) DC were also present in inflamed colonic and ileal mucosa from UC and Crohn's patients but not in noninflamed IBD mucosa or normal mucosa. Expression of the CMRF44 antigen was low on freshly isolated DC, but it was upregulated after 24-h culture on DC from all groups, although significantly less so on DC from UC versus Crohn's or healthy controls (p = 0.024). The CMRF44(+) antigen was mainly associated with CD11c(+) DC, and in UC was inversely related to the Clinical Activity Index (r = -0.69, p = 0.0002). CONCLUSIONS: There is upregulation of costimulatory molecules on blood DC even in very mild IBD but surprisingly, there is divergent expression of the differentiation/activation CMRF44 antigen. Upregulation of costimulatory molecules and divergent expression of CMRF44 in blood DC was also apparent in cGI and aGI but not in non-GI or healthy controls, whereas intestinal CD86(+) and CD40(+) DC were found only in inflamed mucosa from IBD patients. Persistent or distorted activation of blood DC or divergent regulation of costimulatory and activation antigens may have important implications for gut mucosal immunity and inflammation. (Am J Gastroenterol 2001;96:2946-2956. (C) 2001 by Am. Coll. of Gastroenterology).

FAM deubiquitylating enzyme is essential for preimplantation mouse embryo development

FAM is a developmentally regulated substrate-specific deubiquitylating enzyme. It binds the cell adhesion and signalling molecules beta -catenin and A-F-6 in vitro, and stabilises both in mammalian cell culture. To determine if FAM is required at the earliest stages of mouse development we examined its expression and function in preimplantation mouse embryos. FAM is expressed at all stages of preimplantation development from ovulation to implantation. Exposure of two-cell embryos to FAM-specific antisense, but not sense, oligodeoxynucleotides resulted in depletion of the FAM protein and failure Of the embryos to develop to blastocysts. Loss of FAM had two physiological effects, namely, a decrease in cleavage rate and an inhibition of cell adhesive events. Depletion of FAM protein was mirrored by a loss of beta -catenin such that very little of either protein remained following 72 h culture. The residual beta -catenin was localised to sites of cell-cell contact suggesting that the cytoplasmic pool of beta -catenin is stabilised by FAM. Although AF-6 levels initially decreased they returned to normal. However, the nascent protein was mislocalised at the apical surface of blastomeres. Therefore FAM is required for preimplantation mouse embryo development and regulates beta -catenin and AF-6 in vivo. (C) 2001 Elsevier Science Ireland Lid. All rights reserved.

Human pigmentation genes: identification, structure and consequences of polymorphic variation

The synthesis of the visible pigment melanin by the melanocyte cell is the basis of the human pigmentary system, those genes directing the formation, transport and distribution of the specialised melanosome organelle in which melanin accumulates can legitimately be called pigmentation genes. The genes involved in this process have been identified through comparative genomic studies of mouse coat colour mutations and by the molecular characterisation of human hypopigmentary genetic diseases such as OCA1 and OCA2. The melanocyte responds to the peptide hormones a-MSH or ACTH through the MC1R G-protein coupled receptor to stimulate melanin production through induced maturation or switching of melanin type. The pheomelanosome, containing the key enzyme of the pathway tyrosinase, produces light red/yellowish melanin, whereas the eumelanosome produces darker melanins via induction of additional TYRP1, TYRP2, SILV enzymes, and the P-protein. Intramelanosomal pH governed by the P-protein may act as a critical determinant of tyrosinase enzyme activity to control the initial step in melanin synthesis or TYRP complex formation to facilitate melanogenesis and melanosomal maturation. The search for genetic variation in these candidate human pigmentation genes in various human populations has revealed high levels of polymorphism in the MC1R locus, with over 30 variant alleles so far identified. Functional correlation of MC1R alleles with skin and hair colour provides evidence that this receptor molecule is a principle component underlying normal human pigment variation. (C) 2001 Elsevier Science B.V. All rights reserved.

Stable expression of noncytopathic Kunjin replicons simulates both ultrastructural and biochemical characteristics observed during replication of Kunjin virus

This report focuses mainly on the characterization of a Vero cell line stably expressing the flavivirus Kunjin (KUN) replicon C20SDrep (C20SDrepVero). We showed by immunofluorescence and cryoimmunoelectron microscopy that unique flavivirus-induced membrane structures, termed convoluted membranes/paracrystalline structures, were induced in the C20SDrepVero cells. These induced cytoplasmic foci were immunolabeled with KUN virus anti-NS3 antibodies and with antibodies to the cellular markers ERGIC53 (for the intermediate compartment) and protein disulfide isomerase (for the rough endoplasmic reticulum). However, in contrast to the large perinuclear inclusions observed by immunofluorescence with anti-double-stranded (ds)RNA antibodies in KUN virus-infected cells, the dsRNA in C20SDrepVero cells was localized to small isolated foci scattered throughout the cytoplasm, which were coincident with small foci dual-labeled with the trans-Golgi specific marker GaIT. importantly persistent expression of the KUN replicons in cells did not produce cytopathic effects, and the morphology of major host organelles (including Golgi, mitochondria, endoplasmic reticulum, and nucleus) was apparently unaffected. The amounts of plus- and minus-sense RNA synthesis in replicon cells were similar to those in KUN virus-infected cells until near the end of the latent period, but subsequently increases of about 10- and fourfold, respectively, occurred in infected cells. Virus-specified protein synthesis in C20SDrepVero cells was also about 10-fold greater than that in infected cells. When several KUN replicon cell lines were compared with respect to membrane induction, the relative efficiencies increased in parallel with increases in viral RNA and protein synthesis, consistent with the increases observed during the virus infectious cycle. Based on these observations, cell lines expressing less-efficient replicons may provide a useful tool to study early events in flavivirus RNA replication, which are difficult to assess in Virus infections. (C) 2001 Academic press.

Peripheral T-Cell tolerance defined through transgenic mouse studies

Selection in the thymus restricted by MHC and self-peptide shapes the diverse reactivities of the T-cell population which subsequently seeds into the peripheral tissues, in anticipation of the universe of pathogen antigens to which the organism may be exposed. A necessary corollary is the potential for T-cell self-reactivity (autoimmunity) in the periphery. Transgenic mouse models in which transgene expression in the thymus is prevented or excluded, have been particularly useful for determining the immunological outcome when T-cells encounter transgene-encoded 'self' antigen in peripheral tissues. Data suggest that non-mutually exclusive mechanisms of T-cells 'ignoring' self-antigen, T-cell deletion, T-cell anergy and T-cell immunoregulation have evolved to prevent self-reactivity while maintaining T-cell diversity. The peripheral T-cell repertoire, far from being static following maturation through the thymus, is in a dynamic stated determined by these peripheral selective and immunoregulatory influences. This article reviews the evidence with particular reference to CD8+ive T-cells.

Wolbachia-mediated sperm modification is dependent on the host genotype in Drosophila

Estimates of Wolbachia density in the eggs, testes and whole flies of drosophilid hosts have been unable to predict the lack of cytoplasmic incompatibility (CI) expression in so-called mod(-) variants. Consequently, the working hypothesis has been that CI expression, although related to Wolbachia density, is also governed by unknown factors that are influenced by both host and bacterial genomes. Here, we compare the behaviour of the mod(-) over-replicating Wolbachia popcorn strain in its native Drosophila melanogaster host to the same strain transinfected into a novel host, namely Drosophila simulans. We report that (i) the popcorn strain is a close relative of other D. melanogaster infections, (ii) the mod(-) status of popcorn in D. melanogaster appears to result from its inability to colonize sperm bundles, (iii) popcorn is present in the bundles in D. simulans and induces strong CI expression, which demonstrates that the bacterial strain does not lack the genetic machinery for inducing CI and that there is host-species-specific control over Wolbachia tissue tropism, and (iv) infection of sperm bundles by the mod(-) D. simulans wCof strain indicates that there are several independent routes by which a strain can be a CI non-expressor.

Timing and magnitude of peak height velocity and peak tissue velocities for early, average, and late maturing boys and girls

Height, weight, and tissue accrual were determined in 60 male and 53 female adolescents measured annually over six years using standard anthropometry and dual-energy X-ray absorptiometry (DXA). Annual velocities were derived, and the ages and magnitudes of peak height and peak tissue velocities were determined using a cubic spline fit to individual data. Individuals were rank ordered on the basis of sex and age at peak height velocity (PHV) and then divided into quartiles: early (lowest quartile), average (middle two quartiles), and late (highest quartile) maturers. Sex- and maturity-related comparisons in ages and magnitudes of peak height and peak tissue velocities were made. Males reached peak velocities significantly later than females for all tissues and had significantly greater magnitudes at peak. The age at PHV was negatively correlated with the magnitude of PHV in both sexes. At a similar maturity point (age at PHV) there were no differences in weight or fat mass among maturity groups in both sexes. Late maturing males, however, accrued more bone mineral and lean mass and were taller at the age of PHV compared to early maturers. Thus, maturational status (early, average, or late maturity) as indicated by age at PHV is inversely related to the magnitude and late maturers for weight and fat mass in boys and girls. Am. J. Hum. Biol. 13:1-8, 2001. (C) 2001 Wiley-Liss, Inc.

Relationships between crude protein and mineral concentrations in alfalfa and value of alfalfa silage as a mineral source for periparturient cows

One hundred and eight samples from three cultivars of alfalfa were obtained from three cuttings in 1996-1998 to evaluate the relationship between crude protein (CP) and mineral concentrations of alfalfa with cutting and maturation. The CP content drastically decreased from 27.9 to 11.4% on DM with maturity. Highly positive correlations were observed between CP and K in the first and the second cutting of alfalfa. The Ca content remained almost constant throughout the growth period. Four multiparous Holstein cows were assigned an alfalfa silage diet or an orchardgrass silage diet from 3 weeks prepartum to 1 week postpartum to examine the effect on the mineral balance. In the prepartum and postpartum diet, the roughage to concentrate ratio was 70:30 and 50:50, with alfalfa being 50 and 100% of the roughage, respectively. The alfalfa contained 1.93% of K. No metabolic disorders occurred, but the body weight decreased drastically from 1 to 6 days postpartum with each diet because of the high milk production immediately after the parturition. Positive retention of N, Ca, P, Mg, and K was observed prepartum, whereas the cows had negative N and mineral retention from 2 to 4 days postpartum. The Ca and P absorption, and Mg retention of cows with the alfalfa diet were higher than with the grass diet. The plasma Ca and inorganic P were not affected by diet, but the plasma PTH at parturition and plasma hydroxyproline from 1 week prepartum to 1 week postpartum were higher with the alfalfa diet. These results suggest that the low K alfalfa is suitable not only to prevent the incidence of milk fever but also to increase Ca, P and Mg utilization of periparturient cows, but the mineral supplementation is needed for the postpartum cows immediately after the parturition. (C) 2001 Elsevier Science B.V. All rights reserved.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface, We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodo- main of the interleukin-2 alpha (IL-2 alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK1 epithelial cells, Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin, Truncation mutants unable to bind beta -catenin were correctly targeted, showing, contrary to current understanding, that beta -catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine mediated targeting is maintained in UC-PK, cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line, These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

The gene encoding the immunoregulatory signaling molecule CMRF-35A localized to human chromosome 17 in close proximity to other members of the CMRF-35 family

The immunoregulatory signaling (IRS) family includes several molecules, which play major roles in the regulation of the immune response. The CMRF-35A and CMRF-35H molecules are two new members of the IRS family of molecules, that are found on a wide variety of haemopoietic lineages. The extracellular functional interactions of these molecules is presently unknown, although CMRF-35H on initiate an inhibitory signal and is internalized when cross-linked. In this paper, we described the gene structure for the CMRF-35A gene and its localization to human chromosome 17. The gene consists of four exons spanning approximately 4.5 kb. Exon 1 encodes the 5' untranslated region and leader sequence, exon 2 encodes the immunoglobulin (Ig)-like domain, exon 3 encodes the membrane proximal region and exon 4 encodes the transmembrane region, the cytoplasmic tail and the 3' untranslated region. A region in the 5' flanking sequence of the CMRF-35A gene, that promoted expression of a reporter gene was identified. The genes for the CMRF-35A and CMRF-35H molecules are closely linked on chromosome 17. Similarity between the Ig-like exons and the preceding intron of the two genes suggests exon duplication was involved in their evolution. We also identified a further member of the CMRF-35 family, the CMRF-35J pseudogene. This gene appears to have arisen by gene duplication of the CMRF-35A gene. These three loci-the CMRF-35A, CMRF-35J and CMRF-35H genes-form a new complex of IRS genes on chromosome 17.

MUC13, a novel human cell surface mucin expressed by epithelial and hemopoietic cells

Transmembrane mucins are glycoproteins involved in barrier function in epithelial tissues. To identify novel transmembrane mucin genes, we performed a tblastn search of the GenBank(TM) EST data bases with a serine/ threonine-rich search string, and a rodent gene expressed in bone marrow was identified. We determined the cDNA sequence of the human orthologue of this gene, MUC13, which localizes to chromosome band 3q13.3 and generates 3.2-kilobase pair transcripts encoding a 512-amino acid protein comprised of an N-terminal mucin repeat domain, three epidermal growth factor-like sequences, a SEA module, a transmembrane domain, and a cytoplasmic tail (GenBank(TM) accession no. AF286113), MUC13 mRNA is expressed most highly in the large intestine and trachea, and at moderate levels in the kidney, small intestine, appendix, and stomach, In situ hybridization in murine tissues revealed expression in intestinal epithelial and lymphoid cells. Immunohistochemistry demonstrated the human MUC13 protein on the apical membrane of both columnar and goblet cells in the gastrointestinal tract, as well as within goblet cell thecae, indicative of secretion in addition to presence on the cell surface. MUC13 is cleaved, and the beta -subunit containing the cytoplasmic tail undergoes homodimerization, Including MUC13, there are at least five cell surface mucins expressed in the gastrointestinal tract.

Protein targeting to the plasma membrane of adult skeletal muscle fiber: An organized mosaic of functional domains

The plasma membrane of differentiated skeletal muscle fibers comprises the sarcolemma, the transverse (T) tubule network, and the neuromuscular and muscle-tendon junctions. We analyzed the organization of these domains in relation to defined surface markers, beta -dystroglycan, dystrophin, and caveolin-3, These markers were shown to exhibit highly organized arrays along the length of the fiber. Caveolin-3 and beta -dystroglycan/dystrophin showed distinct, but to some extent overlapping, labeling patterns and both markers left transverse tubule openings clear. This labeling pattern revealed microdomains over the entire plasma membrane with the exception of the neuromuscular and muscle-tendon junctions which formed distinct demarcated macrodomains. Our results suggest that the entire plasma membrane of mature muscle comprises a mosaic of T tubule domains together with sareolemmal caveolae and beta -dystroglycan domains. The domains identified with these markers were examined with respect to targeting of viral proteins and other expressed domain-specific markers, We found that each marker protein was targeted to distinct microdomains, The macrodomains were intensely labeled with all our markers. Replacing the cytoplasmic tail of the vesicular stomatitis virus glycoprotein with that of CD4 resulted in retargeting from one domain to another. The domain-specific protein distribution at the muscle cell surface may be generated by targeting pathways requiring specific sorting information but this trafficking is different from the conventional apical-basolateral division. (C) 2001 Academic Press.

A caveolin dominant negative mutant associates with lipid bodies and induces intracellular cholesterol imbalance

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3(DGV) specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1-3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cav(DGV) to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

Flotillin-1-enriched lipid raft domains accumulate on maturing phagosomes

Flotillin-1 was recently shown to be enriched on detergent-resistant domains of the plasma membrane called lipid rafts. These rafts, enriched in sphingolipids and cholesterol, sequester certain proteins while excluding others. Lipid rafts have been implicated in numerous cellular processes including signal transduction, membrane trafficking and molecular sorting. In this study, we demonstrate both morphologically and biochemically that lipid rafts are present on phagosomes, These structures are enriched in flotillin-1 and devoid of the main phagosomes membrane protein lysosomal-associated membrane protein (LAMP1), The flotillin-1 present on phagosomes does not originate from the plasma membrane during phagocytosis but accumulates gradually on maturing phagosomes, Treatment with bafilomycin A1, a compound that inhibits the proton pump ATPase and prevents the fusion of phagosomes with late endocytic organelles, prevents the acquisition of flotillin-1 by phagosomes, indicating that this protein might be recruited on phagosomes from endosomal organelles. A proteomic characterization of the lipid rafts of phagosomes indicates that actin, the alpha- and beta -subunits of heterotrimeric G proteins, as well as subunits of the proton pump V-ATPase are among the constituents of these domains. Remarkably, the intracellular parasite Leishmania donovani can actively inhibit the acquisition of flotillin-1-enriched lipid rafts by phagosomes and the maturation of these organelles. These results indicate that specialized functions required for phagolysosome biogenesis may occur at focal points on the phagosome membrane, and therefore represent a potential target of intracellular pathogens.

Kinase-dependent and kinase-independent functions of EphA4 receptors in major axon tract formation in vivo

The EphA4 receptor tyrosine kinase regulates the formation of the corticospinal tract (CST), a pathway controlling voluntary movements, and of the anterior commissure (AC), connecting the neocortical temporal robes. To study EphA4 kinase signaling in these processes, we generated mice expressing mutant EphA4 receptors either lacking kinase activity or with severely downregulated kinase activity. We demonstrate that EphA4 is required for CST formation as a receptor for which it requires an active kinase domain. In contrast, the formation of the AC is rescued by kinase-dead EphA4, suggesting that in this structure EphA4 acts as a ligand for which its kinase activity is not required. Unexpectedly, the cytoplasmic sterile-alpha motif (SAM) domain is not required for EphA4 functions. Our findings establish both kinase-dependent and kinase-independent functions of EphA4 in the formation of major axon tracts.