Monomeric recombinant TCR ligand reduces relapse rate and severity of experimental autoimmune encephalomyelitis in SJL/J mice through cytokine switch

Our previous studies demonstrated that oligomeric recombinant TCR ligands (RTL) can treat clinical signs of experimental autoimmune encephalomyelitis (EAE) and induce long-term T cell tolerance against encephalitogenic peptides. In the current study, we produced a monomeric I-A(s)/PLP 139-151 peptide construct (RTL401) suitable for use in SJL/J mice that develop relapsing disease after injection of PLP 139-151 peptide in CFA. RTL401 given i.v. or s.c. but not empty RTL400 or free PLP 139-151 peptide prevented relapses and significantly reduced clinical severity of EAE induced by PLP 139-151 peptide in SJL/J or (C57BL/6 x SJL)F(1) mice, but did not inhibit EAE induced by PLP 178-191 or MBP 84-104 peptides in SJL/J mice, or MOG 35-55 peptide in (C57BL/6 x SJL/J)F(1) mice. RTL treatment of EAE caused stable or enhanced T cell proliferation and secretion of IL-10 in the periphery, but reduced secretion of inflammatory cytokines and chemokines. In CNS, there was a modest reduction of inflammatory cells, reduced expression of very late activation Ag-4, lymphocyte function-associated Ag-1, and inflammatory cytokines, chemokines, and chemokine receptors, but enhanced expression of Th2-related factors, IL-10, TGF-beta3, and CCR3. These results suggest that monomeric RTL therapy induces a cytokine switch that curbs the encephalitogenic potential of PLP 139-151-specific T cells without fully preventing their entry into CNS, wherein they reduce the severity of inflammation. This mechanism differs from that observed using oligomeric RTL therapy in other EAE models. These results strongly support the clinical application of this novel class of peptide/MHC class II constructs in patients with multiple sclerosis who have focused T cell responses to known encephalitogenic myelin peptides.     

A Model System for the Study of the Release of Luteinizing Hormone-Releasing Hormone from Isolated Storage Granules

Abstract: We have developed an in vitro system for the study of the release of luteinizing hormone-releasing hormone (LH-RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH-RH-containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH-RH affinity columns. The LH-RH associated with the granules passes freely through the columns, whereas the LH-RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH-RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH-RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40–160 mM. We then studied the effects of pH on the release of LH-RH. Incubation of granules at pH 7.8 in the presence of 160 mM-KC1 resulted in the release from the granules of 14% of the stored LH-RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH-RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH-RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH-RH are stable under defined biochemical conditions, and LH-RH release from these granules is stimulated by ions and MgATP.     

Capillary malformation-arteriovenous malformation, a new clinical and genetic disorder caused by RASA1 mutations

Capillary malformation (CM), or "port-wine stain," is a common cutaneous vascular anomaly that initially appears as a red macular stain that darkens over years. CM also occurs in several combined vascular anomalies that exhibit hypertrophy, such as Sturge-Weber syndrome, Klippel-Trenaunay syndrome, and Parkes Weber syndrome. Occasional familial segregation of CM suggests that there is genetic susceptibility, underscored by the identification of a large locus, CMC1, on chromosome 5q. We used genetic fine mapping with polymorphic markers to reduce the size of the CMC1 locus. A positional candidate gene, RASA1, encoding p120-RasGAP, was screened for mutations in 17 families. Heterozygous inactivating RASA1 mutations were detected in six families manifesting atypical CMs that were multiple, small, round to oval in shape, and pinkish red in color. In addition to CM, either arteriovenous malformation, arteriovenous fistula, or Parkes Weber syndrome was documented in all the families with a mutation. We named this newly identified association caused by RASA1 mutations "CM-AVM," for capillary malformation-arteriovenous malformation. The phenotypic variability can be explained by the involvement of p120-RasGAP in signaling for various growth factor receptors that control proliferation, migration, and survival of several cell types, including vascular endothelial cells.     

Identification of MarvelD3 as a tight junction-associated transmembrane protein of the occludin family

Background

Protein translocation across the membrane of the Endoplasmic Reticulum (ER) is the first step in the biogenesis of secretory and membrane proteins. Proteins enter the ER by the Sec61 translocon, a proteinaceous channel composed of three subunits, α, β and γ. While it is known that Sec61α forms the actual channel, the function of the other two subunits remains to be characterized.

Results

In the present study we have investigated the function of Sec61β in Drosophila melanogaster. We describe its role in the plasma membrane traffic of Gurken, the ligand for the Epidermal Growth Factor (EGF) receptor in the oocyte. Germline clones of the mutant allele of Sec61β show normal translocation of Gurken into the ER and transport to the Golgi complex, but further traffic to the plasma membrane is impeded. The defect in plasma membrane traffic due to absence of Sec61β is specific for Gurken and is not due to a general trafficking defect.

Conclusion

Based on our study we conclude that Sec61β, which is part of the ER protein translocation channel affects a post-ER step during Gurken trafficking to the plasma membrane. We propose an additional role of Sec61β beyond protein translocation into the ER.     

Nickel and cobalt reagents promote selective oxidation of Z-DNA

The structural characteristics of Z-DNA were used to challenge the selectivity of guanine oxidation promoted by nickel and cobalt reagents. Base pairing and stacking within all helical structures studied previously had hindered access to guanine and limited its reaction. However, the Z-helix uniquely retains high exposure of guanine N7. This exposure was sufficient to direct oxidation specifically to a plasmid insert -(CG)(13)AATT(CG)(13)- that adopted a Z-conformation under native supercoiling. An alternative insert -(CG)(7)- retained its B-conformation and demonstrated the expected lack of reactivity. For a nickel salen complex made from a particularly bulky ligand, preferential reaction shifted to the junctions within the Z-DNA insert as is common for large reagents. Inactivation of the nickel reagents by high-salt concentrations prevented parallel investigations of Z-DNA, formed by oligonucleotides. However, the activity of Co(2+) was minimally affected by salt and consequently confirmed the high reactivity of 5'-p(CG)(4) in its Z-conformation. These reagents may now be applied to a broad array of targets, since their structural specificity remains predictable for both complex and helical assemblies of nucleic acids.     

Characterization of B lymphocyte lineage progenitor cells from mice with severe combined immune deficiency disease (SCID) made possible by long term culture

A single gene mutation results in near absence of B and T lymphocytes and their immediate progenitors in mice with severe combined immunodeficiency disease (SCID). However, long term culture conditions allowed rapid outgrowth of lymphocytes from SCID bone marrow suspensions, and this permitted their detailed analysis. The cells were judged to be committed to the B lymphocyte lineage on the basis of expression of the BP-1 antigen, as well as by the density and pattern of expression of other markers. Cultured SCID lymphocytes were indistinguishable from control BALB/c cells in terms of morphology, typing for 13 cell surface markers, and changes in cell surface antigen expression with time in culture. In contrast to cultures of normal cells, which always included IgM synthesizing cells, SCID lymphocytes rarely expressed mu heavy chains. Southern blot analysis demonstrated that at least the first Ig gene rearrangement step had occurred in most of the cultured cells. The patterns of JH gene rearrangements suggested that relatively limited population diversity existed in individual cultures of SCID and normal BALB/c marrow. In addition, there was evidence that abnormal Ig heavy chain gene rearrangements had taken place in lymphocytes from approximately 25% of the SCID cultures. These cells were distinguished by the absence of detectable JH gene segments. kappa light chain genes appeared to be unrearranged in SCID cultured lymphocytes. We conclude that the lymphopoietic microenvironments of SCID mice are probably normal, and the animals have infrequent progenitors of B cells. Aberrant or nonproductive IgH gene rearrangements may account for the absence of pre-B and B cells in SCID mice. This study demonstrates the usefulness of long term culture methodology for isolating rare subsets of non-transformed lymphoid cells from normal and genetically defective hemopoietic tissues.     

Physiological and morphological properties of the metathoracic common inhibitory neuron of the locust

Summary Intracellular recordings were made from the soma of the metathoracic common inhibitory neuron of the locustsSchistocerca andChortoicetes. The soma is passively invaded by a spike of 2–5 mV in amplitude. The response of the common inhibitor to a variety of different inputs was studied. Tests for coupling between the common inhibitory and excitatory motoneurons to the same or antagonistic muscles were made by simultaneous recordings from pairs of neuron somata. No low resistance or synaptically mediated coupling was found. The somata of the two common inhibitory neurons which supply muscles on opposite sides of the body lie together on the ventral surface of the ganglion on the mid-line (Fig. 6). They are not coupled in any way. Cobalt chloride injected into the common inhibitor has shown it to have an extensive and complex dendritic tree confined to the ipsilateral half of the ganglion (Fig. 8). A single branch extends into the mesothoracic ganglion. There are differences in the branching patterns of the dendrites in different animals (Fig. 10).Beit Memorial Research Fellow.     

Inheritance of resistance to neonatal E. coli diarrhoea in the pig: examination of the genetic system

Summary Evidence is presented that a dominant allele, S, is expressed as a receptor for K88 on the brushborder surface of the pig intestinal cell. The homozygous recessive (ss) lacks this receptor. The receptor enables K88 — positive coliforms to adhere to the gut of the piglet which they must do if they are to cause neonatal diarrhoea. The homozygous recessive is thus a disease resistant animal.A possible reason for the persistence of the dominant (susceptible) gene is given.     

Does lethal control of top‐predators release mesopredators? A re‐evaluation of three Australian case studies

Top‐predators can sometimes be important for structuring fauna assemblages in terrestrial ecosystems. Through a complex trophic cascade, the lethal control of top‐predators has been predicted to elicit positive population responses from mesopredators that may in turn increase predation pressure on prey species of concern. In support of this hypothesis, many relevant research papers, opinion pieces and literature reviews identify three particular case studies as supporting evidence for top‐predator control‐induced release of mesopredators in Australia. However, many fundamental details essential for supporting this hypothesis are missing from these case studies, which were each designed to investigate alternative aims. Here, we re‐evaluate the strength of evidence for top‐predator control‐induced mesopredator release from these three studies after comprehensive analyses of associated unpublished correlative and experimental data. Circumstantial evidence alluded to mesopredator releases of either the European Red Fox (Vulpes vulpes) or feral Cat (Felis catus) coinciding with Dingo (Canis lupus dingo) control in each case. Importantly, however, substantial limitations in predator population sampling techniques and/or experimental designs preclude strong assertions about the effect of lethal control on mesopredator populations from these studies. In all cases, multiple confounding factors and plausible alternative explanations for observed changes in predator populations exist. In accord with several critical reviews and a growing body of demonstrated experimental evidence on the subject, we conclude that there is an absence of reliable evidence for top‐predator control‐induced mesopredator release from these three case studies. Well‐designed and executed studies are critical for investigating potential top‐predator control‐induced mesopredator release.