Transfer of central nervous system autoantigens and presentation in secondary lymphoid organs

Dendritic cells are thought to regulate tolerance induction vs immunization by transferring Ags and peripheral signals to draining lymph nodes (LN). However, whether myelin Ag transfer and presentation in LN occurs during demyelinating brain disease is unknown. In this study, we demonstrate redistribution of autoantigens from brain lesions to cervical LN in monkey experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Immunohistochemical analysis revealed significantly more cells containing myelin Ags in cervical LN of monkeys with EAE compared with those of healthy control monkeys. Myelin Ags were observed in cells expressing dendritic cell/macrophage-specific markers, MHC class II, and costimulatory molecules. Moreover, these cells were directly juxtaposed to T cells, suggesting that cognate interactions between myelin-containing APC and T cells are taking place in brain-draining LN. Indeed, myelin Ag-reactive T cells were observed in cervical LN from marmosets and rhesus monkeys. Importantly, these findings were paralleled by our findings in human tissue. We observed significantly more myelin Ag-containing cells in LN of individuals with MS compared with those of control individuals. These cells expressed APC markers, as observed in marmosets and rhesus monkeys. These findings suggest that during MS and EAE, modulation of T cell reactivity against brain-derived Ags also takes place in cervical LN and not necessarily inside the brain. A major implication is that novel therapeutic strategies may be targeted to peripheral events, thereby circumventing the blood-brain barrier.     

The establishment of a network of European human research tissue banks

This is a report of a workshop held on the establishment of human research tissue banking which was held in Levi, Finland 21–24 March 2002.There were 21 participants from 7 European countries. This meeting was attended by representatives from academia, research tissue banks and from the Biotech and Pharmaceutical Industries. The principal aim of the workshop was to find a way to progress the recommendations from ECVAM workshop 44 (ATLA 29, 125–134,2001) and ECVAM workshop 32 (ATLA 26, 763–777, 1998). The workshop represented the first unofficial meeting of the European Network of Research Tissue Banks (ENRTB) steering group. It is expected that in the period preceding the next workshop the ENRTB steering group will co-ordinate the ethical,legislative and organisational aspects of research tissue banking. Key issues dealt with by the Levi workshop included the practical aspects of sharing expertise and experiences across the different European members. Such collaboration between research tissue banks and end users of such material seeks to ultimately enable shared access to human tissue for medical and pharmaco-toxicological research while maintaining strict adherence to differences in legal and ethical aspects related to the use of human tissue in individual countries. This revised version was published online in July 2006 with corrections to the Cover Date.     

A new allele of Gli3 and a new mutation,circletail (Crc), resulting from a single transgenic experiment

While generating transgenic lines, transgene-linked mutations can occur, which are caused by an insertional mutation at a given locus. More rarely, mutations unlinked to the transgene insertion site are observed. In the process of generating a mouse overexpressing the enzyme tyrosinase, we have obtained one transgenic line that appears to carry a semidominant insertional mutation at the Gli3 (extra toes) locus, characterized by polydactyly and skeletal malformations. Additionally, the transgenic line contained a second mutation, Crc (circletail), which appears to be unlinked to the transgene insertion site. Heterozygous Crc mice are incompletely penetrant for a circled-tail phenotype, while all homozygous Crc/Crc mice die at birth of a severe neural tube defect (craniorachischisis). Anatomical evidence from a Crc/Crc; Gli3/+ fetus indicates that these two genes may interact.     

Classification and genetic characterization of pattern-forming Bacilli

One of the more natural but less commonly studied forms of colonial bacterial growth is pattern formation. This type of growth is characterized by bacterial populations behaving in an organized manner to generate readily identifiable geometric and predictable morphologies on solid and semi-solid surfaces. In our first attempt to study the molecular basis of pattern formation in Bacillus subtilis , we stumbled upon an enigma: some strains used to describe pattern formation in B. subtilis did not have the phenotypic or genotypic characteristics of B. subtilis . In this report, we show that these strains are actually not B. subtilis , but belong to a different class of Bacilli , group I. We show further that commonly used laboratory strains of B. subtilis can co-exist as mixed cultures with group I Bacilli , and that the latter go unnoticed when grown on frequently used laboratory substrates. However, when B. subtilis is grown under more stringent semiarid conditions, members of group I emerge in the form of complex patterns. When B. subtilis is grown under less stringent and more motile conditions, B. subtilis forms its own pattern, and members of group I remain unnoticed. These findings have led us to revise some of the mechanistic and evolutionary hypotheses that have been proposed to explain pattern growth in Bacilli .     

Blockade of osteoclast-mediated bone resorption through occupancy of the integrin receptor: A potential approach to the therapy of osteoporosis

Bone resorption requires the tight attachment of the bone-resorbing cells, the osteoclasts, to the bone mineralized matric. Integrins, a class of cell surface adhesion glycoproteins, play a key role in the attachment process. Most integrins bind to their ligands via the arginyl-glycyl-aspartyl (R-G-D) tripeptide present within the ligand sequence. The interaction between integrins and ligaands results in bidirectional transfer of signals across the plasma Membrane. Tyrosine phosphorylaation occurs within cells as a result of integrin binding to ligaands and probably plays a role in the formation of the osteoclast clear zone, a specialized region of the osteoclast membraane maintained by cytoskeletal structure and involved in bone resorption. Human osteoclasts express α₂β₃ and α_vβ₃ integrins on their surface. Such signaling may also lead to “inside-out” effects, like increased expression of integrin receptors on the cell surface, or increased affinity of the integrin to its ligand. The α_vβ₃ integrin, a vitronectin receptor, plays an essential role in bone resorption. Antibodies to this integrin and short synthetic RGD-containing peptides are able to block bone resorption in vitro. Echistatin, an RGD-containing protein from a snake venom, binds to the α_vβ₃ integrin and blocks bone resssorption both in vitro and in vivo. Peptides containing the RGD motif are potential competitive “antagonists” a of the osteoclast integrins and may have utility in the blockage of bone resorption. Agonists may be identified by stimulation of intracellularsignaling. In theory, tissue spacificity can be achieved by (1) introducing specific amino acids in positions adjacent to the RGD sequence, (2) identifying non-RGD integrin binding domains, or (3) modulating the affinity of integrins for their endogenous ligands.     

Hematopoietic, angiogenic and eye defects in Meis1 mutant animals

Meis1 and Hoxa9 expression is upregulated by retroviral integration in murine myeloid leukemias and in human leukemias carrying MLL translocations. Both genes also cooperate to induce leukemia in a mouse leukemia acceleration assay, which can be explained, in part, by their physical interaction with each other as well as the PBX family of homeodomain proteins. Here we show that Meis1-deficient embryos have partially duplicated retinas and smaller lenses than normal. They also fail to produce megakaryocytes, display extensive hemorrhaging, and die by embryonic day 14.5. In addition, Meis1-deficient embryos lack well-formed capillaries, although larger blood vessels are normal. Definitive myeloerythroid lineages are present in the mutant embryos, but the total numbers of colony-forming cells are dramatically reduced. Mutant fetal liver cells also fail to radioprotect lethally irradiated animals and they compete poorly in repopulation assays even though they can repopulate all hematopoietic lineages. These and other studies showing that Meis1 is expressed at high levels in hematopoietic stem cells (HSCs) suggest that Meis1 may also be required for the proliferation/self-renewal of the HSC.     

<Emphasis Type="Italic">CHX10</Emphasis> mutations cause non-syndromic microphthalmia/anophthalmia in Arab and Jewish kindreds

Microphthalmia/anophthalmia is a clinically heterogeneous disorder of eye formation, ranging from small size of a single eye to complete bilateral absence of ocular tissues. The genetic defect underlying isolated autosomal recessive microphthalmia/anophthalmia is yet unclear. We studied four families (two of Arab origin, one of Bedouin origin, and one of Persian-Jewish origin) with autosomal recessive microphthalmia/anophthalmia and no associated eye anomalies, and one Syrian–Jewish family with associated colobomas. Assuming a founder effect in each of the families, we performed homozygosity mapping using polymorphic markers adjacent to human homologues of genes known to be associated with eye absence in various species, namely EYA1, EYA2, EYA3, SIX4, SIX6, PAX6 and CHX10. No association was found with EYA1, EYA2, EYA3, SIX6 or PAX6. In two families, linkage analysis was consistent with possible association with SIX4, but no mutations were found in the coding region of the gene or its flanking intron sequences. In three of the five families, linkage analysis followed by sequencing demonstrated that affected individuals in each family were homozygous for a different CHX10 aberration: a mutation in the CVC domain and a deletion of the homeobox domain were found in two Arab families, and a mutation in the donor-acceptor site in the first intron in the Syrian-Jewish family. There was phenotypic variation between families having different mutations, but no significant phenotypic variation within each family. It has been previously shown that mutations in a particular nucleotide in CHX10 are associated with an autosomal recessive syndrome of microphthalmia/anophthalmia with iris colobomas and cataracts in two families. We now show that different mutations in other domains of the same gene underlie isolated microphthalmia/anophthalmia.     

Modulated fatty acid desaturation via overexpression of two distinct omega-3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco cells and plants

Changes in the degree of fatty acid (FA) desaturation are implicated in plant responses to various abiotic stresses, including heat, salt and drought. However, it is still not known whether decreased levels of linolenic acid, found in many plants subjected to salt and drought stress, reflect a mechanism of defence or damage. We addressed this question by generating tobacco cells and plants ectopically overexpressing two FA desaturases: the cytosolic FAD3 or the plastidic FAD8. A remarkable increase in the ratio of total linolenic to linoleic acids resulted from overexpression of FAD3, whereas ectopic overexpression of FAD8 induced an increased ratio mainly in the plastidic lipids. Here we present evidence that overexpressing FAD8 imposes much greater heat sensitivity than does FAD3 overexpression, in both cultured cells and whole plants. Overexpression of either FAD3 or FAD8 increases tolerance to drought in tobacco plants and to osmotic stress in cultured cells. These findings suggest that a drought-induced decreased level of linolenic acid reflects damage. Our results point to the potential of exploiting FAD overexpression as a tool to ameliorate drought tolerance.     

The BsmI vitamin D receptor gene polymorphism is associated with ulcerative colitis in Jewish Ashkenazi patients

Susceptibility to inflammatory bowel disease (IBD) has a strong genetic component. The vitamin D receptor (VDR) gene maps to a region on chromosome 12 shown to be associated with IBD in some studies. In this case-control study we determined the association between the BsmI VDR gene polymorphism and IBD in patients with Crohn's disease (CD) and ulcerative colits (UC). Three hundred seventy-nine Jewish Israeli patients with IBD, 228 with CD (129 Ashkenazi and 99 non-Ashkenazi), and 151 patients with UC (72 Ashkenazi, 79 non-Ashkenazi) were studied. The control group included 495 healthy blood donors (352 non-Ashkenazi and 143 Ashkenazi). All subjects were genotyped for the BsmI VDR gene polymorphism. The frequency of the BB genotype was higher in Ashkenazi patients with UC compared to Ashkenazi controls (0.21 vs. 0.11, p = 0.042, odds ratio 2.27, 95% confidence interval [CI] 1.06-4.9). There were no differences in the prevalence of the BB genotype or the B allele between ethnically matched patients with CD and UC. Nor were there differences in the BB genotype or B allele frequencies between CD patients and ethnically matched controls. The BsmI VDR gene polymorphism is associated with increased susceptibility to UC in Israeli Ashkenazi patients with UC.     

On the regulatory role of dipeptidyl peptidase IV (=CD=adenosine deaminase complexing protein) on adenosine deaminase activity

The molecular mechanism controlling the variable activity of the malignancy marker adenosine deaminase (ADA) is enigmatic. ADA activity was found to be modulated by the membrane-bound adenosine deaminase complexing protein (CP=DPPIV=CD26). The role of lipid-protein interactions in this modulation was sought. While direct solubilization of ADA in vesicles resulted in loss of ADA activity, the binding of ADA to CP reconstituted in vesicles restored the specific activity. The activity of ADA, free or bound to CP in solution, resulted in continuous linear Arrhenius plots. However, ADA bound to reconstituted CP exhibited two breaks associated with approximately 30% increased activity, at 25 and 13 degrees C, yielding three lines with similar apparent activation energies (E(a)). Continuum solvent model calculations of the free energy of transfer of the transmembrane helix of CP from the aqueous phase into membranes of various widths show that the most favorable orientations of the helix above and below the main phase transition may be different. We suggest that the 20% change in the thickness of the bilayer below and above the main phase transition may modify the orientation of CP in the membrane, thereby affecting substrate accessibility of ADA. This could account for ADA's reduced activity associated with increased membrane fluidity in transformed vs. normal fibroblasts.