High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP-binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on human liver specimens from patients with primary biliary cirrhosis (PBC), chronic hepatitis C virus (HCV) infection, submassive cell necrosis, and normal liver. The expression of MDR1, MDR3, BSEP, MRP1, MRP2, and MRP3 was determined using specific antibodies. Dilution series were constructed to determine the critical staining level in order to estimate the factor of up-regulation. In normal liver, hepatocytes showed canalicular staining for MDR3, BSEP, and MRP2. MDR1 stained the canalicular membrane of hepatocytes as well as that of cholangiocytes. MRP3 showed low immunoreactivity of bile duct epithelial cells and centrilobular hepatocytes only. Normal liver showed no immunoreactivity for MRP1. In diseased liver, the expression of MDR3, BSEP, and MRP2 was relatively stable. In PBC, HCV, and submassive necrosis, the expression levels of MDR1, MRP1, and MRP3 were increased. The strongest immunoreactivity was seen after submassive necrosis, where remaining islands of hepatocytes showed strong canalicular staining for MDR1 and MRP3. Regenerating bile ductules at the interface of portal tracts and necrotic areas stained intensely for MDR1, MRP1, and MRP3. In conclusion, MDR1, MRP1, and MRP3 are up-regulated in hepatocytes in severe human liver disease. Strong MDR1, MRP1, and MRP3 reactivity is seen in regenerating human bile ductules.     

Noradrenergic neuronal development is impaired by mutation of the proneural HASH-1 gene in congenital central hypoventilation syndrome (Ondine's curse)

Congenital central hypoventilation syndrome (CCHS, Ondine's curse) is a rare disorder of the chemical control of breathing. It is frequently associated with a broad spectrum of dysautonomic symptoms, suggesting the involvement of genes widely expressed in the autonomic nervous system. In particular, the HASH-1-PHOX2A-PHOX2B developmental cascade was proposed as a candidate pathway because it controls the development of neurons with a definitive or transient noradrenergic phenotype, upstream from the RET receptor tyrosine kinase and tyrosine hydroxylase. We recently showed that PHOX2B is the major CCHS locus, whose mutation accounts for 60% of cases. We also studied the proneural HASH-1 gene and identified a heterozygous nucleotide substitution in three CCHS patients. To analyze the functional consequences of HASH-1 mutations, we developed an in vitro model of noradrenergic differentiation in neuronal progenitors derived from the mouse vagal neural crest, reproducing in vitro the HASH-PHOX-RET pathway. All HASH-1 mutant alleles impaired noradrenergic neuronal development, when overexpressed from adenoviral constructs. Thus, HASH-1 mutations may contribute to the CCHS phenotype in rare cases, consistent with the view that the abnormal chemical control of breathing observed in CCHS patients is due to the impairment of noradrenergic neurons during early steps of brainstem development.     

Splice variants of the relaxin and INSL3 receptors reveal unanticipated molecular complexity

LGR7 and LGR8 are G protein-coupled receptors that belong to the leucine-rich repeat-containing G-protein coupled receptor (LGR) family, including the thyroid-stimulating hormone (TSH), LH and FSH receptors. LGR7 and LGR8 stimulate cAMP production upon binding of the cognate ligands, relaxin and insulin-like peptide 3 (INSL3), respectively. We cloned several novel splice variants of both LGR7 and LGR8 and analysed the function of four variants. LGR7.1 is a truncated receptor, including only the N-terminal region of the receptor and two leucine rich repeats. In contrast, LGR7.2, LGR7.10 and LGR 8.1 all contain an intact seven transmembrane domain and most of the extracellular region, lacking only one or two exons in the ectodomain. Our analysis demonstrates that although LGR7.10 and LGR8.1 are expressed at the cell surface, LGR7.2 is predominantly retained within cells and LGR7.1 is partially secreted. mRNA expression analysis revealed that several variants are co-expressed in various tissues. None of these variants were able to stimulate cAMP production following relaxin or INSL3 treatment. Unexpectedly, we did not detect any direct specific relaxin or INSL3 binding on any of the splice variants. The large number of receptor splice variants identified suggests an unforeseen complexity in the physiology of this novel hormone-receptor system.     

Profilin-mediated food-induced allergic reactions are associated with oral epithelial remodeling

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Spatial object representation and its use in planning eye movements

The eye movements we make to look at objects require that the spatial information contained in the objects image on the retina be used to generate a motor command. This process is known as sensorimotor transformation and has been generally addressed using simple point targets. Here, we investigate the sensorimotor transformation involved in planning double saccade sequences directed at one or two objects. Using both visually guided saccades toward stationary objects and objects subjected to intrasaccadic displacements, and memory-guided saccades, we found that the coordinate transformations required to program the second saccade were different for saccades aimed at a new target object and saccades that scanned the same object. While saccades aimed at a new object were updated on the basis of the actual eye position, those that scanned the same object were performed with a fixed amplitude, irrespective of the actual eye position. Our findings demonstrate that different abstract representations of space are used in sensory-to-motor transformations, depending on what action is planned on the objects.     

Concentrations of cytokines,soluble interleukin-2 receptor,and soluble CD30 in sera of patients with hepatitis B virus infection during acute and convalescent phases

The immunoregulatory roles of interleukin-2 (IL-2), IL-4, IL-10, gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), the soluble form of the IL-2 receptor (sIL-2R), and the soluble form of CD30 (sCD30) were evaluated in patients with hepatitis B virus (HBV) infection. Two groups of subjects were studied: 15 healthy individuals without hepatitis antecedents and 15 patients with HBV infection. Blood samples were taken during the acute and convalescent phases. The analysis of the samples was done by the enzyme-linked immunosorbent assay technique. IFN-gamma and TNF-alpha levels decreased in the convalescent phase. IL-10, IL-2, and sIL-2R levels increased in the acute and convalescent phases, while sCD30 levels increased during the acute phase. The IL-4 concentrations decreased in both phases. During the acute phase, IFN-gamma and TNF-alpha induced increases in IL-2, sIL-2R, IL-10, and sCD30 levels in serum, which allowed the development of immunity characterized by the nonreactivity of the HBV surface antigen, the onset of antibodies to the HBV surface antigen (anti-HBs), and normal alanine aminotransferase levels during the convalescent phase. Increased IL-2 levels during the acute phase would stimulate the activities of NK cells and CD8(+) lymphocytes, which are responsible for viral clearing. The raised sIL-2R levels reveal activation of T lymphocytes and control of the IL-2-dependent immune response. The sCD30 increment during the acute phase reflects the greater activation of the Th2 cellular phenotype. Its decrease in the convalescent phase points out the decrease in the level of HBV replication. The increase in IL-10 levels could result in a decrease in IL-4 levels and modulate IFN-gamma and TNF-alpha levels during both phases of disease, allowing the maintenance of anti-HBs concentrations.     

Trypanosoma cruzi: Killing and enhanced uptake by resident peritoneal macrophages treated with alpha-2-macroglobulin

We report that alpha-2-macroglobulin (A2M), the physiologically important plasma protease inhibitor and suspected immunomodulator, alters the functional ability of murine resident peritoneal macrophages (RM) to ingest and kill the infective trypomastigote stage ofTrypanosoma cruzi, the aetiological agent of Chagas' disease. Treatment of RM with 500 g/ml A2M for 30 min enhanced the uptake of trypomastigotes, epimastigotes, and amastigotes by 125%, 46%, and 300%, respectively. The same treatment also increased the phagocytosis of sheep erythrocytes opsonized with complement and IgG as well as of galactosylated asialoerythrocytes. After 60–90 min parasite-cell interaction, epi-and amastigotes were killed by the RM, whereas the infection with trypomastigotes was controlled only after 24 h. Other protease inhibitors, bovine serum albumin, and LPS showed no such effect. The production of hydrogen peroxide was not affected by A2M treatment, but the ultrastructural aspects showed trypomastigote damage and enhancement of macrophage membrane ruffling, indicative of macrophage activation. These results suggest that A2M has the ability to modulate, at least functionally, certain receptor-mediated endocytic pathways that, in concert with an activation of possibly oxygen-independent microbicidal mechanisms, could contribute to resistance against the parasite.Abbreviations A2M alpha-2-macroglobulin - F-A2M fast A2M - S-A2M slow A2M - RM resident macrophages - BT bloodstream trypomastigotes - EPI epimastigotes - AMA amastigotes - DMEM Dulbecco's Modified Eagle Medium - PBS phosphate-buffered saline - BSA bovine serum albumin - LPS bacto lipopolysaccharide - STI sovoean trypsin inhibitor - PPA pepstatin A - LPT leupeptin - PNT 1, 10-phenanthroline - TLCK N--tosy-L-lysine-chloromethylketone - E sheep erythrocyte - aE asialoerythrocyte - Gal R receptors for galactosylated particles     

Role of T cell costimulation in anti-viral immunity

Members of both the CD28 and TNFR families can have costimulatory roles in T cell activation. Gene targeted mice as well as in vivo blocking experiments have established distinct roles for CD28/B7; ICOS/ICOSL; CD27/CD70; 4-1BB/4-1BBL and OX40/OX40L during viral infection. Many issues remain to be addressed, including the timing and location of the interactions, the possibility of partial redundancy between related family members and the molecular basis for the specific phenotypes observed in the different gene targeted mice.     

Chimerism studies in HLA-identical nonmyeloablative hematopoietic stem cell transplantation point to the donor CD8(+) T-cell count on day + 14 as a predictor of acute graft-versus-host disease.

Chimerism analysis of hematopoietic cells has emerged as an essential tool in nonmyeloablative hematopoietic stem cell transplantation. We have investigated the development of donor chimerism in granulocytes and CD4(+) and CD8(+) T cells in blood and bone marrow of 24 patients with hematologic malignancies who received HLA-identical sibling peripheral blood stem cell grafts after conditioning with fludarabine and 2 Gy of total body irradiation. The T-cell chimerism of blood and bone marrow was tightly correlated. Complete donor chimerism was reached earlier in the granulocytes than in the T cells. Mixed T-cell chimerism was common at the time of onset of acute graft-versus-host disease (aGVHD), and both CD4(+) and CD8(+) donor T-cell chimerism increased with the occurrence of aGVHD grades II to IV (P =.0002 and P =.019, respectively). The rate of disappearance of recipient CD8(+) T cells was faster in patients with aGVHD grades II to IV than in patients without clinically significant aGVHD (P =.016). This observation indicates a role of graft-versus-lymphohematopoietic tissue reactions in creating complete donor T-cell chimerism. A donor CD8(+) T-cell count above the median on day +14 increased the risk of subsequent development of aGVHD grades II to IV (P =.003).