Immune cells from patients with psoriasis are defective in inducing indoleamine 2,3-dioxygenase expression in response to inflammatory stimuli.

BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an inducible enzyme that suppresses the immune response. The role of IDO as a negative regulator of inflammatory responses has been documented in several experimental autoimmune diseases. OBJECTIVES: To explore the regulation of IDO by immune cells in psoriasis and its relation with disease severity. METHODS: The expression and activity of IDO were assessed by reverse-transcriptase polymerase chain reaction, flow cytometry and high-performance liquid chromatography in peripheral blood of patients with moderate-to-severe plaque-type psoriasis. The ability of immune cells to express IDO in response to inflammatory stimuli was studied. The functional role of IDO expression was evaluated in a regulatory T cell (Treg) differentiation assay, using cocultures of immature monocyte-derived dendritic cells with autologous peripheral CD4+ T cells. RESULTS: Analysis of the kynurenine-to-tryptophan ratio in serum samples indicated higher IDO activity in patients with psoriasis than in healthy controls. However, correlation studies showed lower IDO activity in those patients with higher Psoriasis Area and Severity Index (PASI). Although myeloid dendritic cells from patients with psoriasis expressed higher levels of IDO than those from healthy controls, these cells did not upregulate IDO in response to a combination of tumour necrosis factor-α, interleukin (IL)-1ß and IL-6 cytokines. The defective expression of IDO correlated with PASI. Immature monocyte-derived dendritic cells from patients with psoriasis also expressed low levels of IDO and induced CD4+ Treg differentiation poorly. CONCLUSIONS: Immune cells from patients with psoriasis have a defect in upregulating IDO in response to inflammation associated with the severity of psoriasis.

Apolipoprotein E4 stimulates sulfation of glycosaminoglycans in neural cells.

Apolipoprotein E externally added to neuroblastoma cells in culture stimulates [35S]sulfate incorporation on cell and extracellular matrix glycosaminoglycans (sGAG). This stimulation is mainly observed for ApoE4 compared to ApoE3. The increase in sulfation is not due to increased synthesis as there is no corresponding increase in the [3H]glucosamine incorporation. Since the presence of ApoE is a risk factor for Alzheimer's disease (AD) and the presence of sGAG could facilitate the assembly of the main components, beta-amyloid and tau proteins, of the aberrant structures found in AD, the present study indicates a possible relation between those factors.

Cloning and characterization of Rab and Ran/TC4 cDNA clones in Trichinella spp.

The cloning and characterization of seven Rab and three Ran/TC4 partial cDNA sequences in both cystic (Trichinella spiralis and T. britovi) and noncystic species (T. pseudospiralis) are reported. These molecules were cloned by rapid amplification of cDNA ends via polymerase chain reaction (RACE-PCR), using cDNA from the aforementioned Trichinella spp. coupled to the AP1 adaptor. As primers, AP1 and 5B (derived from the WDTAGQE sequence of region 2 specific for Rab and Ran proteins) sequences were included in the PCR. The cloned cDNAs were sequenced and characterized by both Southern-blot and Northern-blot analysis. Trichinella spp. Rab- and Ran-like molecules showed divergences in both the nucleotide and the deduced amino acid sequences as compared with the corresponding homologues previously described in other organisms. In addition, differences were observed among the Trichinella species, mainly between the cystic and the noncystic species, in both DNA restriction-enzyme polymorphism and expression of the six GTPases isolated.

Expression of IkappaBalpha in the nucleus of human peripheral blood T lymphocytes.

According to current models the inhibitory capacity of I(kappa)B(alpha) would be mediated through the retention of Rel/NF-kappaB proteins in the cytosol. However, I(kappa)B(alpha) has also been detected in the nucleus of cell lines and when overexpressed by transient transfection. To gain better insight into the potential role of nuclear I(kappa)B(alpha) in a physiological context we have analysed its presence in the nucleus of human peripheral blood T lymphocytes (PBL). We demonstrate the nuclear localization of I(kappa)B(alpha) in PBL by different techniques: Western blot, indirect immunofluorescence and electron microscopy. Low levels of nuclear I(kappa)B(alpha) were detected in resting cells whereas a superinduction was obtained after PMA activation. The nuclear pool of I(kappa)B(alpha) showed a higher stability than cytosolic I(kappa)B(alpha) and was partially independent of the resynthesis of the protein. Unexpectedly, the presence of nuclear I(kappa)B(alpha) did not inhibit NF-kappaB binding to DNA and this phenomenon was not due to the presence of IkappaBbeta at the nuclear level. Immunoprecipitation experiments failed to demonstrate an association between nuclear I(kappa)B(alpha) and NF-kappaB proteins. Our results demonstrate that in resting and PMA-activated human PBL, I(kappa)B(alpha) is present in the nucleus in an apparently inactive form unable to disrupt NF-kappaB binding from DNA.

Cloning, sequencing, and expression of the PSA genes from Leishmania infantum.

We describe the molecular cloning of the PSA genes from the Leishmania infantum parasite, which show high sequence similarity with the L. major PSA-2 and L. amazonensis GP46/M2 genes. The PSA genes in L. infantum are arrayed in tandem with a repetition unit of 6 kb. A single-size class of PSA mRNA of 4 kb was detected. The characterised L. infantum PSA genes code for a protein lacking the glycosylphosphatidylinositol addition signal described in other Leishmania species due to the presence of a stop codon located upstream from the DNA sequence coding for the signal. The data obtained after immunoprecipitation of PSA indicate that the protein is present as a water-soluble form, but that also a membrane-anchored form can be detected. The amino acid sequence derived from the isolated PSA gene shows that 60% of the deduced protein is formed by 13 leucine-rich repeats, each one of which is 24 amino acids long. The analysis of the consensus sequence of the repeats revealed that the L. infantum PSA as well as the described L. major PSA-2 and L. amazonensis GP46/M2 proteins may be classified as new members of the leucine-rich repeat-containing protein superfamily. The number of leucine-rich motifs, however, varies considerably between the PSA protein from L. infantum and from the other Leishmania species. The PSA protein is a major antigen determinant during L. infantum infections since 87% of the sera from naturally infected dogs recognise the recombinant PSA purified from Escherichia coli.

Intra-Golgi transport inhibition by megalomicin.

Megalomicin (MGM) is a macrolide antibiotic which has been demonstrated previously to cause an anomalous glycosylation of viral proteins. Here we show that MGM produces profound alterations on Golgi morphology and function. The addition of MGM at 50 microM for 1 h caused a dilation of the Golgi detected by immunofluorescence staining for medial- and trans-Golgi markers. The effect of MGM was clearly more intense on the trans-side of the Golgi, as evidenced in electron microscope preparations. The effect on Golgi morphology was reversible and correlated with an impairment of glycoprotein processing in the trans-Golgi. Thus, although the vesicular stomatitis virus G protein was processed in the presence of MGM to an endoglycosidase H-resistant form, it was poorly sialylated. The sialylation of cellular proteins was also inhibited, resulting in cells with low level of sialylation on the cell surface. However MGM did not inhibit the activities of the galactosyl- or sialyltransferase as measured in vitro. MGM inhibited cis- to medial-, and more strongly, medial- to trans-Golgi transport of vesicular stomatitis virus G protein in an in vitro system, suggesting that the impairment in glycoprotein maturation observed in vivo is the result of intra-Golgi transport inhibition.

The antitumoral compound Kahalalide F acts on cell lysosomes.

The target for the antitumoral peptidic drug, Kahalalide F, has been studied in cultured cells. In the presence of the compound, the cells became impressively swollen, showing the formation of large vacuoles. The formation of these vacuoles appears to be the consequence of changes in lysosomal membranes. Thus, lysosomes are a target for Kahalalide F action.

Trypanosoma cruzi: identification of a membrane cysteine proteinase linked through a GPI anchor.

The biochemical and functional properties of T. cruzi GP50/55, a novel glycosylphosphatidylinositol (GPI)-anchored membrane antigen have been investigated. A 50-52-kDa thiol proteinase activity could be immunoprecipitated with monoclonal antibodies (mAb) directed against GP50/55 (mAb C10), different from the one reactive with mAbs against lysosomal cysteine proteinase GP57/51. Furthermore, the mAb C10-reactive proteinase corresponded to the GPI-anchored surface antigen since the proteolytic and antigenic activity partitioned to the aqueous phase after Triton X114 phase separation of phosphatidylinositol specific phospholipase C (PI-PLC)-treated parasites. Of several proteins immunoprecipitated by a polyclonal anti-lysosomal cysteine proteinase, an mAb to GP57/51 recognized a 60-kDa protein, whereas mAb C10 recognized antigens ranging between 52 and 50 kDa. The GP50/55 antigen detected by mAb C10 is expressed on the parasite surface whereas the GP57/51 antigen is mainly intracellular. The internal peptide sequence obtained from purified GP50/55 showed that it is more homologous to the prototype of the cysteine proteinases superfamily, papain, than to the two T. cruzi lysosomal cysteine proteinases so far described. Our data indicate that the T. cruzi GP50/55 is a novel GPI-anchored cysteine proteinase and may represent another isoform of this heterogeneous group of proteinases.

Association of the regulatory beta-adrenergic receptor kinase with rat liver microsomal membranes.

beta-Adrenergic receptor kinase (beta ARK) is a regulatory enzyme involved in the modulation of beta-adrenergic and other G protein-coupled receptors. It has been described that beta ARK is a cytosolic protein that transiently translocates to the plasma membrane in order to specifically phosphorylate agonist-occupied receptors. In this report, we used beta ARK-specific antibodies to demonstrate that a significant amount of this kinase is present in rat liver microsomal membranes. beta ARK seems to be peripherally associated with the cytosolic side of microsomal membranes since it can be stripped from the membranes by mild salt treatment. Cell-free association experiments indicate that the interaction of beta ARK is reversible, saturable, and strongly inhibited by protease or heat treatment of the microsomes, thus suggesting that beta ARK interacts with a protein component of the microsomal membrane. Gradient fractionation studies indicate that the highest beta ARK-specific activity co-migrates with endoplasmic reticulum enzymatic markers. Furthermore, indirect immunofluorescence and immunogold electron microscopy experiments performed in cultured cells using affinity-purified anti-beta ARK antibodies are consistent with this subcellular localization pattern. Taken together, our data suggest that several beta ARK pools (i.e. microsome-bound, plasma membrane-bound, and cytosolic) may exist inside the cell. Such results are in line with recent reports showing that proteins involved in plasma membrane signal transduction, such as heterotrimeric G proteins, are also associated with membranes of different intracellular organelles.