Abstract

The challenge in trying to understand the pathogenesis of Down syndrome (DS) is to determine how an extra set of approximately 250 normal genes causes the physical and functional abnormalities that collectively constitute the syndrome. And, if this knowledge is to become clinically useful, the challenge is to determine whether there is a limited subset of genes that are the major contributors to the phenotype and, if so, to find how the deleterious effects of over dosage of these genes can be prevented or ameliorated. To this end, a series of genetic models for DS/trisomy 21 have been developed to permit the mapping of various components of the DS phenotype to specific genes or regions of the genome. At the present time, the two most commonly used mouse models for DS are the segmental trisomies Ts65Dn and Ts1Cje, which replicate imbalance of HSA 21q from near the centromere to Mx and from just distal to Sodl to Mx, respectively. Use of these two strains permits a subtractive type of phenotypic mapping to be carried out and constitutes a prototype for future phenotypic mapping using a variety of other segmental trisomies. Our work suggests that the region of Ts65Dn proximal to and including Sodl, which is comprised of abut 30 genes, contains a gene or genes which, when present in three copies, are implicated in the pathogenesis of several specific abnormalities of the trisomic nervous system. (Presented at the 1331st Meeting October 27, 2003.)

Infant mental health in Australia

Campbell Paul

Infant Mental Health sits in a very privileged position. It is at the confluence of developmental psychology, This understanding has now a rapidly expanding basis mental health, psychiatry, paediatrics, child health and children's welfare services. There is now a greater movement for inclusion for families themselves in this field. Donald Winnicott the twentieth century English paediatrician and psychoanalyst has had a major influence on evolution of infant mental health in Australia. Building on major upheavals in societal structures, he encouraged a deep and mindful approach to the care of infants and children. His approach coupled with the natural Australian tendency to question rigid authority has led to a lively and expanding network of a broad range of professionals working with infants and their families. At its very centre is the Baby. Our attempts to understand the baby's own inner perspective is the major driving force in Australian Infant Mental Health. This understanding has now a rapidly expanding basis in the developmental neurosciences. Modern neuroimaging has given us additional insights into the baby's developing brain in the context of her developing world. The baby alone does not exist. This is a necessary "two-brain" set up which is the emerging human. There is much exciting research looking at the linkage of our understanding of the baby's unconscious and inner world with detailed analysis of communicative interaction and attachment systems and mechanisms. Infant mental health, child health and child welfare services need to continue to come together in order to provide that which is best for babies who have the greatest potential and the greatest vulnerability. (Presented at the 1332nd Meeting November 25, 2003.)

Transcriptional profiling of the scleroderma phenotype: insights into sustained fibrosis

Andrew Leask

We have identified mRNAs and proteins overexpressed in scleroderma fibroblasts. These include Connective tissue growth factor (CTGF, CCN2) and endothelin-1. CTGF, a member of the CCN family of proteins, is a cysteine-rich pro-adhesive matricellular protein that plays an essential role in the formation of blood vessels, bone and connective tissue. As expression of this protein is potently induced by transforming growth factor-b (TGFb), it has been hypothesized that CTGF mediates several of the downstream actions of TGFb. In particular, CTGF is pro-fibrotic as CTGF is over-expressed in fibrotic disease, and synergizes with TGFbto promote sustained fibrosis in vivo. Over the last several years, key data regarding the developmental role and structure/function relationship of CTGF have emerged. In addition, increased information concerning the mechanisms underlying the control of CTGF expression in normal and fibrotic cells and the signal transduction pathways through which CTGF acts on cells has been uncovered. The endothelins are a family of endothelium-derived peptides that possess a variety of functions, including vasoconstriction. Addition of endothelin-1 (ET-1) to normal lung fibroblasts induces expression of proteins that promote a contractile phenotype, including a-smooth muscle actin a- SMA). Using specific signal transduction inhibitors, we have probed the pathways through which endothelin promotes fibrosis. Thus, we believe synthesis of excessive CTGF and endothelin greatly contribute to pathological fibrosis in vivo. (Presented at the 1333rd Meeting November 27, 2003.)

The Notch perspective: a role for g-secretase in development and tissue renewal

Hui-Teng Cheng, Yonghua Pan, MeeiHua Lin, Mal-G. Tansey, Brandon Hadland, Stacey Huppert, Xiaolin Tian, Jie Shen, Jeffrey H. Miner and Raphael Kopan

Notch signaling is involved in pronephros development in Xenopus and in glomerulogenesis in mice. However, due to early lethality in mice deficient for some Notch pathway genes and functional redundancy for others, a role for Notch signaling during early stages of metanephric development has not been found. Using an antibody specific to the N-terminal end of g-secretase- cleaved Notch 1, we detected evidence of Notch 1 activation in the comma and S-shaped bodies of the mouse metanephros. We therefore cultured metanephroi in the absence and presence of a g-secretase inhibitor, DAPT, to block Notch signaling. We observed attenuated ureteric bud branching but normal mesenchymal condensation and expression of markers indicating that mesenchyme induction had occurred. Fewer renal epithelial structures were observed, with the most obvious deficiency in proximal tubules and podocytes, which are derived from cells in which activated Notch 1 is normally present. Distal tubulogenesis occurred in reduced numbers, accompanied by an increase in interstitial cells. After a transient three-day exposure to DAPT, a proximal tubules reappeared, but podocyte differentiation failed to recover. These observations suggest that g-secretase activity, probably through Notch, is required for maintaining a competent progenitor as well as for specifying the proximal tubule and podocyte fates. To further dissect the role of Notch 1, the contribution of ES cells deficient in Notch 1 is being analyzed and preliminary results will be described. this work is published: http://www. ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db =PubMed&list_uids=12952904&dopt=Abstract In the second part of my presentation I will present our data regarding the role of Notch signaling in skin development. Several roles have been attributed to Notch signaling in vertebrate skin. Over expression studies demonstrated that Notch activation early disrupts patterning in the chick ectoderm (1-3), its activation late in specific cell types disrupts cell-cell interactions, leading to a non-autonomous phenotype (4). We have mapped activated Notch 1 using cleavage specific antibodies; NICD accumulates in the some matrix and precortical cells and in some outer root sheath and supra-basal epidermal cells. To examine the role of Notch signaling in appendage formation we analyzed the phenotype of mice deficient in Notch 1 or g-secretase using a conditional alleles of Notch 1 or presenilin 1 (PS1). While one allele of PS2 is sufficient for normal skin development, mosaic, conditional removal of both PS1 alleles in a PS2/PS2 background results in patches of naked skin. Histological characterization reveals that g-secretase is not required for patterning or cell fate acquisition in the hair follicle but it was required for maintaining the pilosebaceous unit. Sebaceous glands fail to form, and follicles progressively degenerate from p4 onwards ORS hyperplasia accompanies the progressive degeneration of the hair shaft, leading to formation of epidermal cysts by the start of catagen and loss of discernable dermal papilla. Loss of Notch 1 alone is insufficient to recreate the PS1/PS2 loss, but abnormal follicular development is nonetheless evident.

References
1. Viallet JP, et al: Mechanisms of Development 1998; 72: 159-168
2. Crowe R, Niswander L: Developmental Biology 1998; 195: 70-74
3. Crowe R, Henrique D, Ish-Horowicz D, Niswander L: Development
1998; 125: 767-775
4. Lin M, Leimeister C, Gessler M, Kopan R: Development 2000; 127: 2421-2432

YP, MHL, and RK were supported by NIH PO1AR45254. H.T.C and R.K were supported by NIH GM55479; Alzheimer's Association Grant RG991516, and the Zenith award (RK) ZEN-01-3050. J.H.M. was supported by RO1DK053196 and P50DK045181 and in part by Research Grant 1-FY02-192 from the March of Dimes. Partial support was provided by NIH GM55479 (XT, BH, SSH) and HD044056 (RK, YP). (Presented the 1335th Meeting, November 28, 2003.)

Platelet & endothelial microparticles: new horizons

Lawrence L. Horstman

As indicated by the title, this article explores recent findings suggestive of potentially important new medical applications and research implications of platelet microparticles (PMP) and endothelial microparticles (EMP), rather than rehashing established findings already covered in our reviews of PMP (1999) and EMP (2003). After brief background, Part I emphasizes new discoveries about platelets (immunity, inflammation, angiogenesis, tissue factor, etc) suggestive of important functions of PMP. About six such possible functions are considered, but establishing which of these are indeed physiologically important is a challenge for the future. Assay methods and immediate clinical applications are also covered. Part II focusses mainly on EMP, assay of which is emerging as probably superior to ELISA assay of soluble markers for identifying endothelial dysfunction, for reasons defended. This section includes brief summaries of recent work from our laboratory presented at ASH 2003 (San Diego, CA, Dec 4-10), including the possible relevance of EMP-bound vWF to TTP, role of EMP in transendothelial migration of leukocytes, and application of EMP assay to clinical conditions such as multiple sclerosis, metabolic syndrome, sepsis, and hypercholesterolema. The article concludes with possibilities for future developments such as extending EMP phenotype analysis to discriminate specific types of endothelial injury, and identification of organ-specific locus of injury using peptides from phage display libraries developed at other laboratories. Other kinds of MP, some whose origin is not yet identified, are also considered. In summary, further research may elevate MP analysis beyond its present status as a sideline curiosity, to general recognition as a practical clinical tool. (Presented at the 1337th Meeting December 1, 2003.)

Peripheral B lymphocyte tolerance

Amanda Gavin, Laurent Verkoczy, Djemel Aït-Azzouzene, Annica Mårtensson, Patrick Skog, Bao Duong and David Nemazee

This lecture discusses two interrelated topics, B cell tolerance in the peripheral immune system and BAFF. Using the 3-83 antibody transgenic mouse bred to mice carrying cognate antigen in the liver, we previously found that clonal elimination drastically reduced the precursor frequency of autoreactive cells. The consensus model to explain this tolerance is the 2-signal hypothesis, which proposes that in the absence of T cell help BCR stimulation is a negative signal for B cells. However, this model fails to explain how these same B cells can respond to T-independent type II (TI-2) antigens, raising the question of how they distinguish TI-2 antigens from multimeric self determinants. We propose that B cells use NK-like missing self recognition to provide the needed specificity, as foreign antigens are unlikely to carry self markers. The model has implications for the evolution of the immune system, B lymphocyte signaling, tissue specificity of autoimmunity, and microbial subversion of the immune system. Overexpression of the critical B cell survival cytokine BAFF/ Blys has been associated with autoimmunity. We have discovered a novel splice isoform that regulates BAFF activity and may play a role in limiting B cell activity. The novel form, called DBAFF, is able to heteromultimerize with normal BAFF and can suppress receptor binding and proteolytic release from the cell surface. Preliminary studies from transgenic mice overexpressing wild type or ΔBAFF are consistent with a possible regulatory role for ΔBAFF, raising the possibility that the relative expression levels of BAFF and ΔBAFF regulates tolerance. (Presented at the 1338th Meeting December 12, 2003.)

Clinical applications of recombinant human BMP's: early experience and future development

Thomas A. Einhorn

Nearly forty years ago, Marshall R. Urist made the discovery that the extracellular matrix of bone contains a substance that has the capacity to produce new bone formation when implanted into extracellular sites in a host. This substance, later identified as bone morphogenetic protein (BMP), has become the subject of intense research and development for its therapeutic use in the restoration and treatment of skeletal injuries and conditions. The characterization of molecular clones and activities associated with this protein ultimately led to the recognition of fifteen individual human BMP's. Isolation and expression of complimentary DNA's led to the production of several of these molecules using recombinant gene technology. At this time, two BMP's, BMP-2 and BMP-7 (OP-1) are in clinical use, or are in clinical trials, for their application in the spine, open fractures and tibial nonunions of long bones. Presently, BMP-2, when used in combination with a collagen sponge and in a tapered fusion cage, has been shown to be successful in the treatment of single-level interbody fusions of the lumbar spine. The same molecule was recently shown to reduce the number of secondary interventions in patients with open tibial shaft fractures treated with irrigation and debridement followed by a statically locked intramedullary nail. BMP-7 (OP-1) is currently approved for use in several European countries, Australia and in a limited fashion in the United States. It is indicated for the treatment of recalcitrant nonunions of long bones. Early experience suggests efficacy of these molecules in specific clinical settings. Current research is focused on optimizing the healing capacity of these molecules in patients and developing new indications for their use. (Presented at the 1339th Meeting December 22, 2003.)

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