The extracellular matrices of connective tissues contain growth factors such as transforming growth factor (TGF)-beta1. The possibility arises, therefore, that animal connective tissues that have been excised and rendered acellular in the sterilization, lyophilization, and other preparative processes for human use may still retain active growth factors that could contribute to the clinical efficacy of the product. We therefore analyzed 4M guanidine HCl extracts of a sterilized, acellular matrix, Oasis Wound Matrix, for the presence of TGF-beta1 by a sandwich enzyme-linked immunosorbent assay using the soluble type II receptor for TGF-beta to capture the growth factor, and for biological activity by testing the capacity of the extracts to inhibit 3[H]thymidine incorporation into Mv1Lu cells (mink lung epithelial cells). The enzyme-linked immunosorbent assay determined that TGF-beta1 was present at a concentration of 710.9 +/- 157.7 pg/g dry weight of tissue in Oasis and 768.1 +/- 182.1 pg/g dry weight of tissue in SIS (porcine small intestinal submucosa), the disinfected precursor of Oasis. The growth inhibition assays demonstrated that the Oasis extracts inhibited the proliferation of Mv1Lu cells in culture, consistent with the TGF-beta1 in the material having biological activity. Most of the TGF-beta1 survives the sterilization and lyophilization processes in the preparation of the Oasis Wound Matrix, and is functional in its ability to bind to its receptor and, apparently, in its capacity to inhibit growth.

Recent investigation has shown an interaction between transplanted progenitor cells and resident splenocytes leading to modulation of the immunologic response in neurological injury. We hypothesize that the intravenous injection of multipotent adult progenitor cells (MAPC) confers neurovascular protection after traumatic brain injury through an interaction with resident splenocytes, subsequently leading to preservation of the blood brain barrier. Four groups of rats underwent controlled cortical impact injury (3 groups) or sham injury (1 group). MAPC were injected via the tail vein at two doses (2*106 MAPC/kg or 10*106 MAPC/kg) 2 and 24 hours after injury. Blood brain barrier permeability was assessed by measuring Evans blue dye extravasation (n=6/group). Additionally, splenic mass was measured (n=12/group) followed by splenocyte characterization (n=9/group) including: cell cycle analysis (n=6/group), apoptosis index (n=6/group), cell proliferation (n=6/group), and inflammatory cytokine measurements (n=6/group). Vascular architecture was determined by immunohistochemistry (n=3/group). Traumatic brain injury results in a decrease in splenic mass and increased blood brain barrier permeability. Intravenous infusion of MAPC preserved splenic mass and returned blood brain barrier permeability towards control sham injured levels. Splenocyte characterization indicated an increase in the number and proliferative rate of CD4+ T cells as well as an increase in IL-4 and IL-10 production in stimulated splenocytes isolated from the MAPC treatment groups. Immunohistochemistry demonstrated stabilization of the vascular architecture in the peri-lesion area Traumatic brain injury causes a reduction in splenic mass that correlates with an increase in circulating immune cells leading to increased blood brain barrier permeability. The intravenous injection of MAPC preserves splenic mass and the integrity of the blood brain barrier. Furthermore, the co-localization of transplanted MAPC and resident CD4+ splenocytes is associated with a global increase in IL-4 and IL-10 production and stabilization of the cerebral microvasculature tight junction proteins.

Macrophage-mediated axonal dieback presents an additional challenge to regenerating axons after spinal cord injury. Adult adherent stem cells are known to have immunomodulatory capabilities, but their potential to ameliorate this detrimental inflammation-related process has not been investigated. Using an in vitro model of axonal dieback as well as an adult rat dorsal column crush model of spinal cord injury, we found that multipotent adult progenitor cells (MAPCs) can affect both macrophages and dystrophic neurons simultaneously. MAPCs significantly decrease MMP-9 (matrix metalloproteinase-9) release from macrophages, effectively preventing induction of axonal dieback. MAPCs also induce a shift in macrophages from an M1, or “classically activated” proinflammatory state, to an M2, or “alternatively activated” antiinflammatory state. In addition to these effects on macrophages, MAPCs promote sensory neurite outgrowth, induce sprouting, and further enable axons to overcome the negative effects of macrophages as well as inhibitory proteoglycans in their environment by increasing their intrinsic growth capacity. Our results demonstrate that MAPCs have therapeutic benefits after spinal cord injury and provide specific evidence that adult stem cells exert positive immunomodulatory and neurotrophic influences.

Some polio vaccines prepared from 1954 to 1961 were contaminated with infectious SV40. It has been assumed that all polio vaccines were SV40 free in the United States after 1961 and in other countries after 1962. Following a WHO requirement that was prompted by the detection of SV40 in some human tumors, we conducted a multilaboratory study to test for SV40 polio vaccines prepared after 1961. Vaccine samples from 13 countries and the WHO seed were initially tested by PCR. The possible presence of intact and/or infectious SV40 DNA in PCR-positive samples was tested by transfection and infection of permissive CV-1 cells. All results were verified by immunohistochemistry, cloning, and sequencing. All the vaccines were SV40 free, except for vaccines from a major eastern European manufacturer that contained infectious SV40. We determined that the procedure used by this manufacturer to inactivate SV40 in oral poliovirus vaccine seed stocks based on heat inactivation in the presence of MgCl 2 did not completely inactivate SV40. These SV40-contaminated vaccines were produced from early 1960s to about 1978 and were used throughout the world. Our findings underscore the potential risks of using primary monkey cells for preparing poliovirus vaccines, because of the possible contamination with SV40 or other monkey viruses, and emphasize the importance of using well-characterized cell substrates that are free from adventitious agents. Moreover, our results indicate possible geographic differences in SV40 exposure and offer a possible explanation for the different percentage of SV40-positive tumors detected in some laboratories. (Cancer Res 2005; 65(22): 10273-9)

Only a fraction of subjects exposed to asbestos develop malignant mesothelioma (MM), suggesting that additional factors may render some individuals more susceptible. We tested the hypothesis that asbestos and Simian virus (SV40) are cocarcinogens. Asbestos and SV40 in combination had a costimulatory effect in inducing ERK1͞2 phosphorylation and activator protein-1 (AP-1) activity in both primary Syrian hamster mesothelial cells (SHM) and primary human mesothelial cells (HM). Ap-1 activity caused the expression and activation of matrix metalloprotease (MMP)-1 and MMP-9, which in turn led to cell invasion. Experiments using siRNA and chemical inhibitors confirmed the specificity of these results. The same effects were observed in HM and SHM. Experiments in hamsters showed strong cocarcinogenesis between asbestos and SV40: SV40 did not cause MM, asbestos caused MM in 20% of hamsters, and asbestos and SV40 together caused MM in 90% of hamsters. Significantly lower amounts of asbestos were sufficient to cause MM in animals infected with SV40. Our results indicate that mineral fibers and viruses can be cocarcinogens and suggest that lower amounts of asbestos may be sufficient to cause MM in individuals infected with SV40.activator protein-1 ͉ ERK1͞2 ͉ matrix metalloprotease ͉ environmental carcinogenesis ͉ viral oncology M alignant mesothelioma (MM) is a malignancy of the mesothelial cells that form the serosal membranes that cover the chest and abdominal cavities. Median survival is Ϸ12 months, and MM causes 2,000-3,000 deaths per year in the USA and Ϸ1,000 deaths per year in the U.K (1). The continuing increase in the incidence of MM has been associated to the widespread use of asbestos in the past century. The mechanisms of asbestos carcinogenesis have been linked to the extracellular response regulated kinase ERK1͞2 and activator protein-1 (AP-1) pathways (2), to the activation of proinflammatory cytokines and NFB (3), and to the production of reactive mutagenic oxygen species by nearby lung macrophages exposed to asbestos (4).Only a fraction (Ϸ5%) of subjects exposed to high levels of asbestos develop MM. This finding suggests that additional factors, such as SV40 infection and genetic predisposition, may render some individuals more susceptible to asbestos carcinogenicity (1, 5). SV40 is a monkey DNA virus that induces MM in hamsters (6). Following this observation, numerous laboratories have detected SV40 in MM biopsies although the prevalence of SV40 varied in different studies from Ϸ6% to 60%, and some studies did not detect SV40 (7-11). SV40 contaminated human polio vaccines worldwide from 1955 until Ϸ1961. Epidemiological studies comparing cohorts born before or after 1961 detected an increased relative risk of 3 for MM in pre-1961 cohorts that included many individuals vaccinated with contaminated polio vaccines (12). However, the overall epidemiological evidence was considered inconclusive because of differences in ages among the cohorts studied and because it was uncertain that cohorts born after 1961 we...