United States regulatory requirements for skin and eye irritation testing.

PURPOSE: Eye and skin irritation test data are required or considered by chemical regulation authorities in the United States to develop product hazard labelling and/or to assess risks for exposure to skin- and eye-irritating chemicals. The combination of animal welfare concerns and interest in implementing methods with greater human relevance has led to the development of non-animal skin- and eye-irritation test methods. To identify opportunities for regulatory uses of non-animal replacements for skin and eye irritation tests, the needs and uses for these types of test data at U.S. regulatory and research agencies must first be clarified. METHODS: We surveyed regulatory and non-regulatory testing needs of U.S. Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) agencies for skin and eye irritation testing data. Information reviewed includes the type of skin and eye irritation data required by each agency and the associated decision context: hazard classification, potency classification, or risk assessment; the preferred tests; and whether alternative or non-animal tests are acceptable. Information on the specific information needed from non-animal test methods also was collected. RESULTS: A common theme across U.S. agencies is the willingness to consider non-animal or alternative test methods. Sponsors are encouraged to consult with the relevant agency in designing their testing program to discuss the use and acceptance of alternative methods for local skin and eye irritation testing. CONCLUSIONS: To advance the implementation of alternative testing methods, a dialog on the confidence of these methods to protect public health and the environment must be undertaken at all levels.

Mesenchymal stem cell therapy for acute radiation syndrome: innovative medical approaches in military medicine.

After a radiological or nuclear event, acute radiation syndrome (ARS) will present complex medical challenges that could involve the treatment of hundreds to thousands of patients. Current medical doctrine is based on limited clinical data and remains inadequate. Efforts to develop medical innovations that address ARS complications are unlikely to be generated by industry because of market uncertainties specific to this type of injury. A prospective strategy could be the integration of cellular therapy to meet the medical demands of ARS. The most clinically advanced cellular therapy to date is the administration of mesenchymal stem cells (MSCs). Results of currently published investigations describing MSC safety and efficacy in a variety of injury and disease models demonstrate the unique qualities of this reparative cell population in adapting to the specific requirements of the damaged tissue in which the cells integrate. This report puts forward a rationale for the further evaluation of MSC therapy to address the current unmet medical needs of ARS. We propose that the exploration of this novel therapy for the treatment of the multivariate complications of ARS could be of invaluable benefit to military medicine.

Serial cardiac magnetic resonance imaging of injected mesenchymal stem cells.

BACKGROUND: Delivery and tracking of endomyocardial stem cells are limited by the inability to image transplanted cells noninvasively in the beating heart. We hypothesized that mesenchymal stem cells (MSCs) could be labeled with a iron fluorophore particle (IFP) to provide MRI contrast in vivo to assess immediate and long-term localization. METHODS AND RESULTS: MSCs were isolated from swine. Short-term incubation of MSCs with IFP resulted in dose-dependent and efficient labeling. Labeled cells remained viable for multiple passages and retained in vitro proliferation and differentiation capacity. Labeled MSCs (10(4) to 10(6) cells/150 microL) were injected percutaneously into normal and freshly infarcted myocardium in swine. One, 3, and 1 animals underwent serial cardiac MRI (1.5T) for 4, 8, and 21 days, respectively. MRI contrast properties were measured both in vivo and in vitro for cells embedded in agar. Injection sites containing as few as 10(5) MSCs could be detected and contained intact IFP-bearing MSCs on histology. CONCLUSIONS: IFP labeling of MSCs imparts useful MRI contrast, enabling ready detection in the beating heart on a conventional cardiac MR scanner after transplantation into normal and infarcted myocardium. The dual-labeled MSCs can be identified at locations corresponding to injection sites, both ex vivo using fluorescence microscopy and in vivo using susceptibility contrast on MRI. This technology may permit effective in vivo study of stem cell retention, engraftment, and migration.

Highly efficient endosomal labeling of progenitor and stem cells with large magnetic particles allows magnetic resonance imaging of single cells.

Tracking transplanted stem cells using magnetic resonance imaging (MRI) could offer biologic insight into homing and engraftment. Ultrasmall dextran-coated iron oxide particles have previously been developed for uptake into cells to allow MRI tracking. We describe a new application of much larger, micron-scale, iron oxide magnetic particles with enhanced MR susceptibility, which enables detection of single cells at resolutions that can be achieved in vivo. In addition, these larger particles possess a fluorophore for histologic confirmation of cell distribution. We demonstrate highly efficient, nontoxic, endosomal uptake of these particles into hematopoietic CD34+ cells and mesenchymal stem cells documented by confocal and electron microscopy. Labeled cells retain biologic activity with preservation of colony-forming ability and differentiation capacity. MRI studies could detect labeled CD34+ cells and mesenchymal stem cells (MSCs) at single cell resolution. This appears to be a promising tool for serial noninvasive monitoring of in vivo cell homing and localization using MRI.

A gene encoding a novel anti-adhesive protein is expressed in growing cells and restricted to anterior-like cells during development of Dictyostelium.

The Dictyostelium gene ampA, initially identified by the D11 cDNA, encodes a novel anti-adhesive-like protein. The ampA gene product inhibits premature cell agglutination during growth and modulates cell-cell and cell-substrate adhesion during development. Analysis of the promoter indicates that cap site-proximal sequence directs ampA expression during both growth and early development. Expression following tip formation is controlled by more distal sequence, which contains TTGA repeats known to regulate prestalk cell gene expression in other promoters. Comparison of reporter gene expression and endogenous mRNA accumulation indicates that during growth the ampA gene is expressed in an increasing number of cells as a function of density. The number of cells expressing the ampA gene drops as development initiates, but the cells that continue to express the gene do so at high levels. These cells are initially scattered throughout the entire aggregate. By the tip formation stage, however, the majority of ampA-expressing cells are localized to the mound periphery, with only a few cells remaining scattered in the upper portion of the mound. In the final culminant, ampA is expressed only in the upper cup, lower cup, and basal disc. Although reporter expression is observed in cells that migrate anteriorly to a banded region just posterior to the tip, expression is rarely observed in the extreme tip. AmpA protein however, is localized to the tip as well as to ALCs during late development. The results presented here suggest that ampA gene expression is shut off in ALCs that continue along the prestalk differentiation pathway before they are added to the primordial stalk.

A novel disintegrin domain protein affects early cell type specification and pattern formation in Dictyostelium.

The cellular slime mold, Dictyostelium discoideum is a non-metazoan organism, yet we now demonstrate that a disintegrin domain-containing protein, the product of the ampA gene, plays a role in cell type specification. Disintegrin domain-containing proteins are involved in Notch signaling in Drosophila and C. elegans via an ectodomain shedding mechanism that depends on a metalloprotease domain. The Dictyostelium protein lacks a metalloprotease domain. Nonetheless, analysis of cell type specific reporter gene expression during development of the ampA null strain identifies patterning defects that define two distinct roles for the AmpA protein in specifying cell fate. In the absence of a functional ampA gene, cells prematurely specify as prespore cells. Prestalk cell differentiation and migration are delayed. Both of these defects can be rescued by the inclusion of 10% wild-type cells in the developing null mutant aggregates, indicating that the defect is non-cell autonomous. The ampA gene is also demonstrated to be necessary in a cell-autonomous manner for the correct localization of anterior-like cells to the upper cup of the fruiting body. When derived from ampA null cells, the anterior-like cells are unable to localize to positions in the interior of the developing mounds. Wild-type cells can rescue defects in morphogenesis by substituting for null cells when they differentiate as anterior-like cells, but they cannot rescue the ability of ampA null cells to fill this role. Thus, in spite of its simpler structure, the Dictyostelium ampA protein carries out the same diversity of functions that have been observed for the ADAM and ADAMTS families in metazoans.

A novel Dictyostelium gene encoding multiple repeats of adhesion inhibitor-like domains has effects on cell-cell and cell-substrate adhesion.

The Dictyostelium protein AmpA (adhesion modulation protein A) is encoded by the gene originally identified by the D11 cDNA clone. AmpA contains repeated domains homologous to a variety of proteins that influence cell adhesion. The protein accumulates during development, reaching a maximal level at the finger stage. Much of the AmpA protein is found extracellularly during development, and in culminants, AmpA is found in association with anterior-like cells. Characterization of an ampA- strain generated by gene replacement reveals a significant increase in cell-cell clumping when cells are starved in nonnutrient buffer suspensions. Developing ampA- cells are also more adhesive to the underlying substrate and are delayed in developmental progression, with the severity of the delay increasing as cells are grown in the presence of bacteria or on tissue culture dishes rather than in suspension culture. Reintroduction of the ampA gene rescues the developmental defects of ampA- cells; however, expression of additional copies of the gene in wild-type cells results in more severe developmental delays and decreased clumping in suspension culture. We propose that the AmpA protein functions as an anti-adhesive to limit cell-cell and cell-substrate adhesion during development and thus facilitates cell migration during morphogenesis.

AmpA, a modular protein containing disintegrin and ornatin domains, has multiple effects on cell adhesion and cell fate specification.

Proteins containing disintegrin domains play a variety of roles in regulating processes involving adhesion, migration and cell type specification during development of many metazoan organisms. Most disintegrin domain containing proteins belong to the ADAM (a disintegrin and a metalloprotease) family of proteins that also contain a metalloprotease domain. Here we describe a small secreted protein from Dictyostelium that contains multiple repeated domains sharing homology with both the disintegrin motif and with a second class of fibrinogen receptor antagonists, the ornatins. This protein, called AmpA for its role in modulating adhesion, differs from the ADAM family proteins in that it lacks a metalloprotease domain. Nonetheless, it appears to be involved in the same complex spectrum of developmental functions as the metazoan ADAM family proteins. Here we review the structure and evolution of this protein and its function in cell adhesion and cell type specification. We discuss possible mechanisms by which it might function and review the emerging evidence for a close coupling between cell adhesion and cell type specification.