Kinetics of gene expression in murine cutaneous graft-versus-host disease

The kinetics of gene expression associated with the development of cutaneous graft-versus-host disease (GVHD) were examined in a mouse model of MHC-matched allogeneic hematopoietic stem cell transplantation. Ear skin was obtained from recipient mice with or without GVHD between 7 and 40 days after transplantation for histopathological analysis and gene expression profiling. Gene expression patterns were consistent with early infiltration and activation of CD8(+) T and mast cells, followed by CD4(+) T, natural killer, and myeloid cells. The sequential infiltration and activation of effector cells correlated with the histopathological development of cutaneous GVHD and was accompanied by up-regulated expression of many chemokines and their receptors (CXCL-1, -2, -9, and -10; CCL-2, -5, -6, -7, -8, -9, -11, and -19; CCR-1 and CCR-5), adhesion molecules (ICAM-1, CD18, Ly69, PSGL-1, VCAM-1), molecules involved in antigen processing and presentation (TAP1 and TAP2, MHC class I and II, CD80), regulators of apoptosis (granzyme B, caspase 7, Bak1, Bax, and BclII), interferon-inducible genes (STAT1, IRF-1, IIGP, GTPI, IGTP, Ifi202A), stimulators of fibroblast proliferation and matrix synthesis (interleukin-1beta, transforming growth factor-beta1), and markers of keratinocyte proliferation (keratins 5 and 6), and differentiation (small proline-rich proteins 2E and 1B). Many acute-phase proteins were up-regulated early in murine cutaneous GVHD including serum amyloid A2 (SAA2), SAA3, serpins a3g and a3n, secretory leukocyte protease inhibitor, and metallothioneins 1 and 2. The kinetics of gene expression were consistent with the evolution of cutaneous pathology as well as with current models of disease progression during cutaneous GVHD.     

Representation of heading direction in far and near head space

Manipulation of objects around the head requires an accurate and stable internal representation of their locations in space, also during movements such as that of the eye or head. For far space, the representation of visual stimuli for goal-directed arm movements relies on retinal updating, if eye movements are involved. Recent neurophysiological studies led us to infer that a transformation of visual space from retinocentric to a head-centric representation may be involved for visual objects in close proximity to the head. The first aim of this study was to investigate if there is indeed such a representation for remembered visual targets of goal-directed arm movements. Participants had to point toward an initially foveated central target after an intervening saccade. Participants made errors that reflect a bias in the visuomotor transformation that depends on eye displacement rather than any head-centred variable. The second issue addressed was if pointing toward the centre of a wide-field expanding motion pattern involves a retinal updating mechanism or a transformation to a head-centric map and if that process is distance dependent. The same pattern of pointing errors in relation to gaze displacement was found independent of depth. We conclude that for goal-directed arm movements, representation of the remembered visual targets is updated in a retinal frame, a mechanism that is actively used regardless of target distance, stimulus characteristics or the requirements of the task.     

Inhibition of platelet adherence to brain microvasculature protects against severe Plasmodium berghei malaria

Some patients with Plasmodium falciparum infections develop cerebral malaria, acute respiratory distress, and shock and ultimately die even though drug therapy has eliminated the parasite from the blood, suggesting that a systemic inflammatory response contributes to malarial pathogenesis. Plasmodium berghei-infected mice are a well-recognized model of severe malaria (experimental severe malaria [ESM]), and infected mice exhibit a systemic inflammatory response. Because platelets are proposed to contribute to ESM and other systemic inflammatory responses, we determined whether platelet adherence contributes to experimental malarial pathogenesis. Indeed, a significant (P < 0.005) increase in the number of rolling and adherent platelets was observed by intravital microscopy in brain venules of P. berghei-infected mice compared with the number in uninfected controls. P-selectin- or ICAM-1-deficient mice exhibit increased survival after P. berghei infection. We observed a significant (P < 0.0001) reduction in the morbidity of mice injected with anti-CD41 (alpha(IIb) or gpIIb) monoclonal antibody on day 1 of P. berghei infection compared with the morbidity of infected controls injected with rat immunoglobulin G. Additionally, platelet rolling and adhesion in brain venules were reduced in P. berghei mice lacking either P-selectin or ICAM-1 or when the platelets were coated with anti-CD41 monoclonal antibody. Unlike other inflammatory conditions, we did not detect platelet-leukocyte interactions during P. berghei malaria. Because (i). leukocyte adhesion is not markedly altered in the absence of P-selectin or ICAM-1 and (ii). CD41 is not an adhesion molecule for parasitized erythrocytes, these findings support the hypothesis that inhibition of platelet adhesion to the brain microvasculature protects against development of malarial pathogenesis.     

Autoantibodies against mannose-binding lectin in systemic lupus erythematosus

In systemic lupus erythematosus (SLE), autoantibodies directed against complement components of the classical pathway, especially against C1q, are associated with severe disease and are of prognostic value for flares of lupus nephritis. Mannose-binding lectin (MBL), the recognition unit of the MBL pathway of complement activation, has structural similarities to C1q. Deficiencies of MBL have been shown to predispose to the development of SLE and to influence the course of the disease. We hypothesized that the presence of autoantibodies to MBL, analogous to autoantibodies to C1q in patients with SLE, may contribute to disease development. The occurrence of anti-MBL autoantibodies was assessed by enzyme-linked immunosorbent assay (ELISA) of 68 serum samples from 20 patients with SLE and in serum from 70 healthy controls. Levels of antibodies directed against MBL were significantly higher in patients with SLE compared to healthy subjects. No significant difference was found between patients with active disease compared to those with inactive disease. While the occurrence of anti-C1q autoantibodies was associated with renal involvement, no such relationship was found for anti-MBL autoantibodies. A significant correlation was found between anti-MBL and anti-C1q antibody levels. The level of anti-MBL antibodies was negatively correlated with MBL-complex activity of circulating MBL. Anti-MBL autoantibodies were of the immunoglobulin G (IgG) isotype and the binding site of IgG anti-MBL was located in the F(ab')2 portion. We conclude that anti-MBL are present in sera from SLE patients and influence the functional activity of MBL.     

Phenotype delineation of ZNF462 related syndrome

Zinc finger protein 462 (ZNF462) is a relatively newly discovered vertebrate specific protein with known critical roles in embryonic development in animal models. Two case reports and a case series study have described the phenotype of 10 individuals with ZNF462 loss of function variants. Herein, we present 14 new individuals with loss of function variants to the previous studies to delineate the syndrome of loss of function in ZNF462. Collectively, these 24 individuals present with recurring phenotypes that define a multiple congenital anomaly syndrome. Most have some form of developmental delay (79%) and a minority has autism spectrum disorder (33%). Characteristic facial features include ptosis (83%), down slanting palpebral fissures (58%), exaggerated Cupid's bow/wide philtrum (54%), and arched eyebrows (50%). Metopic ridging or craniosynostosis was found in a third of study participants and feeding problems in half. Other phenotype characteristics include dysgenesis of the corpus callosum in 25% of individuals, hypotonia in half, and structural heart defects in 21%. Using facial analysis technology, a computer algorithm applying deep learning was able to accurately differentiate individuals with ZNF462 loss of function variants from individuals with Noonan syndrome and healthy controls. In summary, we describe a multiple congenital anomaly syndrome associated with haploinsufficiency of ZNF462 that has distinct clinical characteristics and facial features.     

Lack of correlation between in vivo peroxidatic activity patterns and tumoricidal activity in vitro of peritoneal macrophages after intraperitoneal immunization with tumor cells

Immunization of C57BL mice with one inoculum of 10(7) DBA/2-derived SL2 lymphosarcoma cells resulted in a +/- 20-fold increase in the total number of peritoneal cells. The number of macrophages showed a 10-fold increase from 3 x 10(6) (control mice) to 3.4 x 10(7) cells at day 8 after immunization. Within this macrophage population, four different cell types, based on the ultrastructural peroxidatic activity patterns, could be distinguished: exudate macrophages, resident macrophages, resident-exudate macrophages and peroxidatic-activity-negative macrophages. The number of exudate macrophages significantly increased in the peritoneal cavity after immunization: at day 8 after immunization, a peak value of 10(7) cells was observed. At the same time, there were 2.2 x 10(7) peroxidase-activity-negative macrophages present (representing the control value x 50). Significant in vitro tumoricidal activity of the isolated macrophages could not be measured until 8 days after immunization. At that time, a cytotoxicity index of 68 was reached. After immunization of the C57BL mice with 3 injections with allogeneic SL2 cells, there were no dramatic changes in the number of peritoneal cells after the last immunization. Only immediately after the last immunization was a minor increase in peroxidatic-activity-negative macrophages seen. But already at 5 days after the last immunization, the composition of the peritoneal suspension was similar to that of non-immunized mice with predominantly resident macrophages. The cytotoxicity of the peritoneal macrophages from hyperimmunized mice was constantly high during 1-15 days after the last immunization (cytotoxicity index ranged from 66-72). In order to study which type(s) of macrophage(s) (resident, exudate, resident-exudate or peroxidatic-activity-negative) is/are responsible for the cytotoxicity measured in vitro, peritoneal cell suspensions (obtained after immunization) were fractionated according to their affinity to wheat germ agglutinin (WGA) coupled to Sepharose columns. Comparison of the values of cytotoxicity measured before and after separation into "subtypes" of the macrophages revealed that the expression of cytotoxicity is not correlated with any of the "sub-types", especially when the peroxidatic activity pattern is is taken as a criterion.     

The rational design of TAP inhibitors using peptide substrate modifications and peptidomimetics

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) translocates peptides from the cytosol into the lumen of the endoplasmic reticulum. This step precedes the binding of peptides to MHC class I molecules and is essential for cell surface expression of the MHC class I/peptide complex. TAP has a broad sequence specificity and a preference for peptides of around 9 amino acids. To synthesize inhibitors for TAP, we studied various alterations of the peptide substrate. The results indicate that TAP is stereospecific and that peptide bonds engineered into isosteric structures can improve translocation of the peptide. Furthermore, TAP is able to translocate peptides with large side chains that correspond to a peptide of ～ 21 amino acids in extended conformation. Peptides with longer side chains compete for the peptide binding site of TAP but fail to be translocated. Therefore, they represent the first rationally designed inhibitors of TAP.     

Functional characterization of a sialyltransferase-deficient mutant of Neisseria gonorrhoeae.

Previous studies indicate that sialylation of lipopolysaccharide (LPS) by host CMP-N-acetylneuraminic acid (CMP-NANA) catalyzed by bacterial sialyltransferase rendered gonococci resistant to killing by phagocytes, to entry into epithelial cell lines, to killing by immune serum and complement, and to absorption of complement component C3. These results have been confirmed by comparing a sialyltransferase-deficient mutant (strain JB1) with its parent (strain F62) in appropriate tests. In contrast to F62, JB1 was very susceptible to killing by human polymorphonuclear phagocytes in opsonophagocytosis tests and incubation with CMP-NANA did not decrease the level of killing. The inherent resistance of F62 in these tests was probably due to LPS sialylation by CMP-NANA and lactate present in the phagocytes. A JB1 variant expressing the invasion-associated Opa protein was as able to enter Chang human conjunctiva epithelial cells as an Opa-positive variant of F62, suggesting that the sialyltransferase is not required for Opa-mediated entry. After incubation with CMP-NANA, the number of F62 variant gonococci entering cells but not that of JB1 variant gonococci was drastically reduced. Both JB1 and F62 were killed by incubation with rabbit antibody to gonococcal major outer membrane protein, protein I, and human complement, but only F62 was rendered resistant to the killing by incubation with CMP-NANA. Finally, both JB1 and F62 absorbed similar amounts of complement component C3 and the binding was decreased by incubation with CMP-NANA only for the wild type, F62.