Multistate Outbreak of Salmonella Virchow Infections Linked to a Powdered Meal Replacement Product-United States, 2015-2016.

Background: Nontyphoidal Salmonella is the leading cause of bacterial gastroenteritis in the United States. Meal replacement products containing raw and "superfood" ingredients have gained increasing popularity among consumers in recent years. In January 2016, we investigated a multistate outbreak of infections with a novel strain of Salmonella Virchow. Methods: Cases were defined using molecular subtyping procedures. Commonly reported exposures were compared with responses from healthy people interviewed in the 2006-2007 FoodNet Population Survey. Firm inspections and product traceback and testing were performed. Results: Thirty-five cases from 24 states were identified; 6 hospitalizations and no deaths were reported. Thirty-one of 33 (94%) ill people interviewed reported consuming a powdered supplement in the week before illness; of these, 30 (97%) reported consuming product A, a raw organic powdered shake product consumed as a meal replacement. Laboratory testing isolated the outbreak strain of Salmonella Virchow from leftover product A collected from ill people's homes, organic moringa leaf powder (an ingredient in product A), and finished product retained by the firm. Firm inspections at 3 facilities linked to product A production did not reveal contamination at the facilities. Traceback investigation identified that the contaminated moringa leaf powder was imported from South Africa. Conclusions: This investigation identified a novel outbreak vehicle and highlighted the potential risk with similar products not intended to be cooked by consumers before consuming. The company issued a voluntary recall of all implicated products. As this product has a long shelf life, the recall likely prevented additional illnesses.

A simple method for using silicone elastomer masks for quantitative analysis of cell migration without cellular damage or substrate disruption.

Cell migration is fundamental to many biological processes, including development, normal tissue remodeling, wound healing, and many pathologies. However, cell migration is a complex process, and understanding its regulation in health and disease requires the ability to manipulate and measure this process quantitatively under controlled conditions. This report describes a simple in vitro assay for quantitative analysis of cell migration in two-dimensional cultures that is an inexpensive alternative to the classic "scratch" assay. The method described utilizes flexible silicone masks fabricated in the lab according to the research demands of the specific experiment to create a cell-free area for cells to invade, followed by quantitative analysis based on widely available microscopic imaging tools. This experimental approach has the important advantage of visualizing cell migration in the absence of the cellular damage and disruption of the substrate that occurs when the "wound" is created in the scratch assay. This approach allows the researcher to study the intrinsic migratory characteristics of cells in the absence of potentially confounding contributions from cellular responses to injury and disruption of cell-substrate interactions. This assay has been used with vascular smooth muscle cells, fibroblasts, and epithelial cell types, but should be applicable to the study of practically any type of cultured cell. Furthermore, this method can be easily adapted for use with fluorescence microscopy, molecular biological, or pharmacological manipulations to explore the molecular mechanisms of cell migration, live cell imaging, fluorescence microscopy, and correlative immunolabeling.

MMP-2 expression by fibroblasts is suppressed by the myofibroblast phenotype.

During wound healing, fibroblasts transition from quiescence to a migratory state, then to a contractile myofibroblast state associated with wound closure. We found that the myofibroblast phenotype, characterized by the expression of high levels of contractile proteins, suppresses the expression of the pro-migratory gene, MMP-2. Fibroblasts cultured in a 3-D collagen lattice and allowed to develop tension showed increased contractile protein expression and decreased MMP-2 levels in comparison to a stress-released lattice. In 2-D cultures, factors that promote fibroblast contractility, including serum or TGF-ß, down-regulated MMP-2. Pharmacologically inducing F-actin disassembly or reduced contractility increased MMP-2 expression, while conditions that promote F-actin assembly suppressed MMP-2 expression. In all cases, changes in MMP-2 levels were inversely related to changes in the contractile marker, smooth muscle α-actin. To determine if the mechanisms involved in contractile protein gene expression play a direct role in MMP-2 regulation, we used RNAi-mediated knock-down of the myocardin-like factors, MRTF-A and MRTF-B, which induced the down-regulation of contractile protein genes by fibroblasts under both serum-containing and serum-free conditions. In the presence of serum or TGF-ß, MRTF-A/B knock-down resulted in the up-regulation of MMP-2; serum-free conditions prevented this increased expression. Together, these results indicate that, while MMP-2 expression is suppressed by F-actin formation, its up-regulation is not simply a consequence of contractile protein down-regulation.

TGF-beta suppresses the upregulation of MMP-2 by vascular smooth muscle cells in response to PDGF-BB.

During platelet-derived growth factor (PDGF)-BB-mediated recruitment to neovascular sprouts, vascular smooth muscle cells (VSMCs) dedifferentiate from a contractile to a migratory phenotype. This involves the downregulation of contractile markers such as smooth muscle (SM) alpha-actin and the upregulation of promigration genes such as matrix metalloproteinase (MMP)-2. The regulation of MMP-2 in response to PDGF-BB is complex and involves both stimulatory and inhibitory signaling pathways, resulting in a significant delay in upregulation. Here, we provide evidence that the delay in MMP-2 upregulation may be due to the autocrine expression and activation of transforming growth factor (TGF)-beta, which is known to promote the contractile phenotype in VSMCs. Whereas PDGF-BB could induce the loss of stress fibers and focal adhesions, TGF-beta was able to block or reverse this transition to a noncontractile state. TGF-beta did not, however, suppress early signaling events stimulated by PDGF-BB. Over time, though PDGF-BB induced increased TGF-beta1 levels, it suppressed TGF-beta2 and TGF-beta3 expression, leading to a net decrease in the total TGF-beta pool, resulting in the upregulation of MMP-2. Together, these findings indicate that MMP-2 expression is suppressed by a threshold level of active TGF-beta, which in turn promotes a contractile VSMC phenotype that prevents the upregulation of MMP-2.

Matrix metalloproteinase-2 expression by vascular smooth muscle cells is mediated by both stimulatory and inhibitory signals in response to growth factors.

In response to growth factors, vascular smooth muscle cells (VSMCs) undergo a phenotypic modulation from a contractile, non-proliferative state to an activated, migratory state. This transition is characterized by changes in their gene expression profile, particularly by a significant down-regulation of contractile proteins. Platelet-derived growth factor (PDGF)-BB has long been known to initiate VSMC de-differentiation and mitogenesis. Insulin-like growth factor (IGF)-I, on the other hand, has differing effects depending on the model studied. Here, we report that both IGF-I and PDGF-BB stimulated VSMC de-differentiation of rat heart-derived SMCs in culture, although only PDGF-BB was capable of inducing proliferation. Although both PDGF-BB and IGF-I stimulation resulted in decreased smooth muscle alpha-actin expression and increased matrix metalloproteinase (MMP)-2 expression, the response to IGF-I was significantly more rapid. The increased MMP-2 expression in response to both growth factors was due to increased transcription rates and was dependent on the action of phosphatidylinositol 3-kinase (PI3K) and its downstream effector, Akt. Both PDGF-BB and IGF-I activated PI3K/Akt to similar degrees; however, only PDGF-BB concomitantly stimulated an inhibitory signaling pathway that antagonized the effects of Akt but did not alter the extent or duration of Akt activation. Together, these findings suggest that changes in MMP-2 expression are part of the program of VSMC phenotypic modulation and that both PDGF-BB and IGF-I, despite their different abilities to induce proliferation in this model, are capable of inducing VSMC activation.

Transcriptional and posttranscriptional regulation of angiopoietin-2 expression mediated by IGF and PDGF in vascular smooth muscle cells.

Angiopoietins play a significant role in vascular development and angiogenesis. Both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) bind the receptor tyrosine kinase Tie2. However, while Ang1 signaling results in the stabilization of vessel structure, Ang2 has been linked to vascular instability. The ratio of these two Tie2 ligands is thus critical for vascular stability and remodeling. This study identifies a mechanism of growth factor-mediated reduction in Ang2 expression in vascular smooth muscle cells (VSMCs). In response to PDGF, VSMCs downregulated Ang2 mRNA levels by 75% within 4 h, with a subsequent decrease in Ang2 protein levels. Quantitation of endogenous transcription rates revealed that PDGF stimulation did not alter Ang2 transcription rates, but instead induced a posttranscriptional mechanism of rapid Ang2 mRNA destabilization. The Ang2 mRNA half-life was reduced by at least 50% after PDGF treatment. The PDGF-induced mRNA turnover mechanism was dependent on several MAPK pathways, including ERK and JNK. In contrast, IGF-I, which did not significantly activate ERK or JNK, stimulated increased Ang2 expression through transcriptional activation. These findings demonstrate that VSMCs adjust Ang2 expression through multiple mechanisms, including changes in transcription as well as posttranscriptional mRNA destabilization.

Matrix metalloproteinases and the ontogeny of scarless repair: the other side of the wound healing balance.

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process. In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3. Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.