A phase 3, open-label, controlled, randomized, multicenter trial evaluating the efficacy and safety of StrataGraft® construct in patients with deep partial-thickness thermal burns.

Objective: This phase 3 study evaluated StrataGraft construct as a donor-site sparing alternative to autograft in patients with deep partial-thickness (DPT) burns.

Methods: Patients aged ≥18 years with 3-49% total body surface area (TBSA) thermal burns were enrolled. In each patient, 2 DPT areas (≤2000cm total) of comparable depth after excision were randomized to either cryopreserved StrataGraft or autograft.

An open-label, prospective, randomized, controlled, multicenter, phase 1b study of StrataGraft skin tissue versus autografting in patients with deep partial-thickness thermal burns.

Objective: This open-label, controlled, randomized study assessed the safety, tolerability, and efficacy of StrataGraft tissue compared to autograft in the treatment of deep partial-thickness (DPT) burns.

Methods: Thirty subjects with DPT thermal burns (3%-43% total body surface area) were treated with StrataGraft tissue as follows: cohort 1, ≤220 cm refrigerated tissue; cohort 2, ≤440 cm refrigerated tissue; and cohort 3, ≤440 cm cryopreserved tissue. On each subject, two comparable areas of DPT burn were randomized to receive StrataGraft tissue or autograft.

Clinical Evaluation of NIKS-Based Bioengineered Skin Substitute Tissue in Complex Skin Defects: Phase I/IIa Clinical Trial Results.

Background: Complex skin defects, such as burns and acute cutaneous trauma, are life-threatening injuries, often requiring temporary allograft placement to maintain fluid homeostasis and prevent infection until permanent wound closure is possible.

The Problem: The current standard of care for the management of full-thickness wounds that are unable to be closed in a single surgical stage is temporary coverage with cadaver allograft until an acceptable wound bed has been established. This approach has limitations including limited availability of human cadaver skin, the risk of disease transmission from cadaveric grafts, and inconsistent cadaver allograft quality.

StrataGraft skin substitute is well-tolerated and is not acutely immunogenic in patients with traumatic wounds: results from a prospective, randomized, controlled dose escalation trial.

Objective: The goal of this study was to assess the immunogenicity and antigenicity of StrataGraft skin tissue in a randomized phase I/II clinical trial for the temporary management of full-thickness skin loss.

Background: StrataGraft skin tissue consists of a dermal equivalent containing human dermal fibroblasts and a fully stratified, biologically active epidermis derived from Near-diploid Immortalized Keratinocyte S (NIKS) cells, a pathogen-free, long-lived, consistent, human keratinocyte progenitor.

Methods: Traumatic skin wounds often require temporary allograft coverage to stabilize the wound bed until autografting is possible.

Erythrocyte scaffolding protein p55/MPP1 functions as an essential regulator of neutrophil polarity.

As mediators of innate immunity, neutrophils respond to chemoattractants by adopting a highly polarized morphology. Efficient chemotaxis requires the formation of one prominent pseudopod at the cell front characterized by actin polymerization, while local inhibition suppresses the formation of rear and lateral protrusions. This asymmetric control of signaling pathways is required for directional migration along a chemotactic gradient.

Calpain inhibition impairs TNF-alpha-mediated neutrophil adhesion, arrest and oxidative burst.

Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in many chronic inflammatory disorders, including rheumatoid arthritis, and contribute to recruitment of neutrophils into areas of inflammation. TNF-alpha induces a stop signal that promotes neutrophil firm adhesion and inhibits neutrophil polarization and chemotaxis. Calpain is a calcium-dependent protease that mediates cytoskeletal reorganization during cell migration.

Selective and tunable gradient device for cell culture and chemotaxis study.

This article describes a microfluidic device for cell culture and chemotaxis studies under various temporal and spatial concentration gradients of the medium or chemoattractant. Vertical membranes formed using in situ fabrication are employed to avoid fluid flow inside the cell observation chamber. Thus, the medium and chemoattractants are primarily provided by diffusion, maintaining cell-cell communication via secreted factors.

Phase I/II clinical evaluation of StrataGraft: a consistent, pathogen-free human skin substitute.

Background: Large wounds often require temporary allograft placement to optimize the wound bed and prevent infection until permanent closure is feasible. We developed and clinically tested a second-generation living human skin substitute (StrataGraft). StrataGraft provides both a dermis and a fully-stratified, biologically-functional epidermis generated from a pathogen-free, long-lived human keratinocyte progenitor cell line, Neonatal Immortalized KeratinocyteS (NIKS).

Modulation of neutrophil function by a secreted mucinase of Escherichia coli O157:H7.

Escherichia coli O157:H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157:H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins.

Neutrophil dysfunction in a family with a SAPHO syndrome-like phenotype.

SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, osteitis) is an inflammatory disorder of the bone, skin, and joints. We describe a family with multiple affected members who segregate a SAPHO syndrome-like phenotype, and we report the results of neutrophil studies and candidate gene analysis. We obtained written informed consent and a family history and reviewed medical records.

A platform for assessing chemotactic migration within a spatiotemporally defined 3D microenvironment.

While the quantification of cell movement within defined biochemical gradients is now possible with microfluidic approaches, translating this capability to biologically relevant three-dimensional microenvironments remains a challenge. We introduce an accessible platform, requiring only standard tools (e.g.

Analysis of neutrophil polarization and chemotaxis.

Neutrophil polarization and directed migration (chemotaxis) are critical for the inflammatory response. Neutrophil chemotaxis is achieved by the sensing of narrow gradients of chemoattractant and the subsequent polarization and directed migration toward the chemotactic source. Despite recent progress, the signaling mechanisms that regulate neutrophil polarization during chemotaxis have not been clearly defined.

Type Igamma PIP kinase is a novel uropod component that regulates rear retraction during neutrophil chemotaxis.

Cell polarization is necessary for directed migration and leukocyte recruitment to inflamed tissues. Recent progress has been made in defining the molecular mechanisms that regulate chemoattractant-induced cell polarity during chemotaxis, including the contribution of phosphoinositide 3-kinase (PI3K)-dependent phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P(3)] synthesis at the leading edge. However, less is known about the molecular composition of the cell rear and how the uropod functions during cell motility.

Analysis of neutrophil chemotaxis.

Neutrophils are the initial responders to bacterial infection or other inflammatory stimuli and comprise a key component of the innate immune response. In addition to their unique morphology and antimicrobial activity, neutrophils are characterized by the ability to migrate rapidly up shallow gradients of attractants in vivo. The directed migration of neutrophils, referred to as chemotaxis, requires the temporal and spatial regulation of intracellular signaling pathways allowing the neutrophil to detect a gradient of attractant, polarize, and migrate rapidly toward the highest concentration of the chemoattractant.

Characterization of a membrane-based gradient generator for use in cell-signaling studies.

This paper describes a method to create stable chemical gradients without requiring fluid flow. The absence of fluid flow makes this device amenable to cell signaling applications where soluble factors can impact cell behavior. This device consists of a membrane-covered source region and a large volume sink region connected by a microfluidic channel.

Neutrophil chemotaxis in a patient with neonatal-onset multisystem inflammatory disease and Muckle-Wells syndrome.

Background: Neonatal-onset multisystem inflammatory disease (NOMID)/chronic infantile neurologic, cutaneous, and articular syndrome is an autoinflammatory disease characterized by urticarial rash, arthropathy, and central nervous system inflammation.

Objective: To describe a 13-year-old girl with overlapping symptoms of NOMID and Muckle-Wells syndrome who has a mutation in cryopyrin (NALP3).

Methods: We examined neutrophil migration using transwell assay and time-lapse videomicroscopy.

TNF-alpha promotes a stop signal that inhibits neutrophil polarization and migration via a p38 MAPK pathway.

Neutrophils are a major component of the inflammatory response in patients with asthma and other inflammatory conditions. Proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-alpha), are increased in the airway of patients with severe asthma and have been implicated in the recruitment of neutrophils into areas of inflammation. Here, we show that TNF-alpha induces a stop signal that promotes firm neutrophil adhesion and inhibits neutrophil polarization and chemotaxis to chemoattractants including interleukin-8 and C5a.

Calpain regulates neutrophil chemotaxis.

Cell polarization is required for directed cell migration. We investigated the role of the calcium-dependent protease calpain during neutrophil chemotaxis and found that calpain inhibition induced neutrophil adhesion, polarization, and rapid chemokinesis in the absence of exogenous activators. Resting neutrophils display constitutive calpain activity with mu-calpain being the predominant active isoform.

Identification of an additional isoform of STAT5 expressed in immature macrophages.

We are interested in understanding the molecular basis of macrophage (Mphi) differentiation and activation by cytokines. Recent reports have suggested that the transcription factor STAT5 may play a role in Mphi differentiation. In the experiments described here, we assessed the expression of STAT5-related molecules in three Mphi cell lines, RAW 264.

Analysis of the IFN-gamma-signaling pathway in macrophages at different stages of maturation.

We previously demonstrated that the macrophage cell lines RAW 264.7 and WEHI-3 exhibit distinct patterns of gene expression in response to IFN-gamma. This difference is controlled at the transcriptional level and results from a specific inability of the less mature WEHI-3 cells to utilize either the IFN-stimulated response element or the gamma-activated sequence DNA regulatory element in response to stimulation with IFN-gamma, while other aspects of IFN-gamma gene induction remain intact.

Mechanisms of murine RANTES chemokine gene induction by Newcastle disease virus.

We have previously defined the lipopolysaccharide (LPS)-responsive element (LRE) in the promoters of murine RANTES (regulated on activation normal T-cell expressed) (MuRantes) and murine IP-10/crg-2, chemokines which have potent chemotactic properties for inflammatory cells including monocytes and T lymphocytes. In the present work, we studied the transcriptional mechanism of MuRantes gene induction by virus and compared it with that of LPS in an effort to understand the host responses to virus and bacterial toxins at the molecular level. MuRantes mRNA expression is induced by Newcastle disease virus (NDV) and LPS in the RAW 264.