Mary A Lokuta

Publications (21) View all

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  Available from: Mary A Lokuta

  Article: 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.

  John M Centanni, Joely A Straseski, April Wicks, Jacquelyn A Hank, Cathy A Rasmussen, Mary A Lokuta, Michael J Schurr, Kevin N Foster, Lee D Faucher, Daniel M Caruso, Allen R Comer, B Lynn Allen-Hoffmann
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  ABSTRACT: 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. 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. Traumatic skin wounds often require temporary allograft coverage to stabilize the wound bed until autografting is possible. StrataGraft and cadaveric allograft were placed side by side on 15 patients with full-thickness skin defects for 1 week before autografting. Allografts were removed from the wound bed and examined for allogeneic immune responses. Immunohistochemistry and indirect immunofluorescence were used to assess tissue structure and cellular composition of allografts. In vitro lymphocyte proliferation assays, chromium-release assays, and development of antibodies were used to examine allogeneic responses. One week after patient exposure to allografts, there were no differences in the numbers of T or B lymphocytes or Langerhans cells present in StrataGraft skin substitute compared to cadaver allograft, the standard of care. Importantly, exposure to StrataGraft skin substitute did not induce the proliferation of patient peripheral blood mononuclear cells to NIKS keratinocytes or enhance cell-mediated lysis of NIKS keratinocytes in vitro. Similarly, no evidence of antibody generation targeted to the NIKS keratinocytes was seen. These findings indicate that StrataGraft tissue is well-tolerated and not acutely immunogenic in patients with traumatic skin wounds. Notably, exposure to StrataGraft did not increase patient sensitivity toward or elicit immune responses against the NIKS keratinocytes. We envision that this novel skin tissue technology will be widely used to facilitate the healing of traumatic cutaneous wounds.This study was registered at www.clinicaltrials.gov (NCT00618839).
  Annals of surgery 04/2011; 253(4):672-83. · 7.90 Impact Factor
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  Available from: Mary A Lokuta

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

  Brendan J Quinn, Emily J Welch, Anthony C Kim, Mary A Lokuta, Anna Huttenlocher, Anwar A Khan, Shafi M Kuchay, Athar H Chishti
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  ABSTRACT: 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. Here, we identify the MAGUK protein p55/MPP1 as a mediator of the frontness signal required for neutrophil polarization. We developed a p55 knockout (p55(-/-)) mouse model, and demonstrate that p55(-/-) neutrophils form multiple transient pseudopods upon chemotactic stimulation, and do not migrate efficiently in vitro. Upon agonist stimulation, p55 is rapidly recruited to the leading edge of neutrophils in mice and humans. Total F-actin polymerization, along with Rac1 and RhoA activation, appear to be normal in p55(-/-) neutrophils. Importantly, phosphorylation of Akt is significantly decreased in p55(-/-) neutrophils upon chemotactic stimulation. The activity of immunoprecipitated phosphatidylinositol 3-kinase gamma (PI3Kgamma), responsible for chemoattractant-induced synthesis of PIP(3) and Akt phosphorylation, is unperturbed in p55(-/-) neutrophils. Although the total amount of PIP(3) is normal in p55(-/-) neutrophils, PIP(3) is diffusely localized and forms punctate aggregates in activated p55(-/-) neutrophils, as compared to its accumulation at the leading edge membrane in the wild type neutrophils. Together, these results show that p55 is required for neutrophil polarization by regulating Akt phosphorylation through a mechanism that is independent of PI3Kgamma activity.
  Proceedings of the National Academy of Sciences 11/2009; 106(47):19842-7. · 9.68 Impact Factor
• Chapter: Analysis of Neutrophil Chemotaxis

  Paul A. Nuzzi, Mary A. Lokuta, Anna Huttenlocher
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  ABSTRACT: 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. A challenge to understanding neutrophil chemotaxis is the inherent difficulty encountered when working with primary neutrophils, which are difficult to purify in the resting state, are not easily transfected, are terminally differentiated, and have a short life span after purification. Here we discuss neutrophil purification methods and chemotaxis assays and provide methodology for working with a neutrophil-like cell line, the HL-60 promyelocytic leukemia cell line. We also discuss methods for HL-60 transfection using retroviral approaches and chemotaxis assays used with differentiated HL-60 cells. Key WordsChemotaxis–neutrophil–HL-60 cell line–time-lapse video microscopy–Transwell assay
  02/2008: pages 23-35;
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  Available from: Mary A Lokuta

  Article: Calpain regulates neutrophil chemotaxis.

  M A Lokuta, P A Nuzzi, A Huttenlocher
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  ABSTRACT: 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. Our findings suggest that constitutive calpain activity in resting neutrophils may function as a negative regulator of protrusion and migration. Specific inhibition of mu-calpain, but not m-calpain, induced neutrophil polarization and chemokinesis. In contrast to IL-8-induced chemokinesis, the chemokinesis induced by calpain inhibition was not reduced in the presence of pertussis toxin, suggesting that calpain functions downstream of G protein-coupled receptors. Further, both calpain inhibition and stimulation with IL-8 and formyl-Met-Leu-Phe (fMLP) induced an increase in Cdc42 and Rac activation. These findings are consistent with the involvement of calpain in chemotaxis pathways. Accordingly, calpain inhibition decreased neutrophil chemotaxis and directional persistence in a gradient of IL-8 and fMLP. Together, these data reveal a previously uncharacterized function for calpain in neutrophils and suggest that localized modulation of calpain activity may regulate neutrophil chemotaxis downstream of G-protein-coupled receptors.
  Proceedings of the National Academy of Sciences 05/2003; 100(7):4006-11. · 9.68 Impact Factor
• Article: Identification of an additional isoform of STAT5 expressed in immature macrophages.

  M A Lokuta, M A McDowell, D M Paulnock
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  ABSTRACT: 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 264.7, WEHI-3, and WEHI-3D+, which represent different stages of Mphi maturation, and also in primary peritoneal and bone marrow Mphi from BALB/c mice. The studies revealed that the previously characterized STAT5a and STAT5b isoforms are detectable at both the mRNA and protein levels in these Mphi populations. Additional STAT5-related proteins were detected by immunoblot analysis and were preferentially expressed in both the immature WEHI-3 cell population and the adherent bone marrow population containing immature Mphi. These results identify new isoforms of STAT5 and demonstrate that distinct patterns of expression of STAT5-related proteins are observed in Mphi at different stages of maturation.
  The Journal of Immunology 09/1998; 161(4):1594-7. · 5.79 Impact Factor