John N. Vournakis

Comparison of poly-N-acetyl glucosamine (P-GlcNAc) with absorbable collagen (Actifoam), and fibrin sealant (Bolheal) for achieving hemostasis in a swine model of splenic hemorrhage.

OBJECTIVE To compare the hemostatic capabilities of poly-Nacetylglucosamine (p-GlcNAc) with three currently available products: Actifoam, Surgicel, and Bolheal fibrin glue. This study was conducted in a controlled animal model, with monitoring of hematologic parameters over the course of the study. Two series were conducted, one in unheparinized animals comparing Bolheal fibrin sealant, Actifoam (absorbable collagen, AC), and Surgicel (ORC) with p-GlcNAc, and the second in systemically heparinized animals comparing p-GlcNAc with AC. METHODS This study was performed in immature female Yorkshire White swine. Splenic lacerations controlled for length and depth of wound were used as sources of bleeding, with one material used per wound to assess hemostatic effectiveness. A total of 97 wounds in 12 animals were created for the study, 74 wounds in unheparinized animals, and 23 wounds in the heparinized animals. In the heparinized animals, hemostatic efficacy was judged by number of applications needed to achieve complete hemostasis. In the unheparinized animals, hemostatic efficacy was judged by length of time required to achieve complete hemostasis (p-GlcNAc vs. fibrin sealant) or the number of applications needed to achieve complete hemostasis (p-GlcNAc vs. AC or ORC). RESULTS In systemically heparinized animals, p-GlcNAc demonstrated greater hemostatic efficacy (72.7 %) in one application than did the control material (0%), p < 0.01. In the unheparinized animals, p-GlcNAc took less time to achieve hemostasis (mean, 22.9 seconds) than fibrin sealant (mean, 172.9 seconds), p < 0.01. p-GlcNAc achieved hemostasis with a greater efficacy (79.2%) in one application than did the AC or ORC (16.7%), p < 0.01, whereas there was no difference in the efficacy of the control materials. CONCLUSION The results of the previous series in unheparinized animals demonstrated that p-GlcNAc in the form of a membrane is a more effective topical hemostatic agent than Bolheal fibrin glue, AC or ORC. The results in the anticoagulated animals similarly demonstrate that p-GlcNAc is a more effective topical hemostatic agent than the control material AC. These data indicate that p-GlcNAc is a promising hemostatic agent as evaluated in this model.

A pilot study evaluating the efficacy of a fully acetylated poly-N-acetyl glucosamine membrane formulation as a topical hemostatic agent ☆ ☆☆

BACKGROUND Topical hemostatic agents are frequently needed for control of intraoperative bleeding. Currently available topical products each have potential drawbacks, making a more effective topical hemostatic agent desirable. This study was performed to evaluate the effectiveness of a particular formulation of a newly available polysaccharide polymer, poly-N-acetyl glucosamine (p-GlcNAc), as a topical hemostatic agent for use in the operating room. Swine splenic incision and splenic capsular stripping hemorrhage models were initially used, with a subsequent pilot human study then performed. METHODS For the swine splenic incision model, anesthetized immature female Yorkshire white swine had a 3 x 8 mm incision created on the spleen. One of 3 agents (p-GlcNAc membrane, oxidized cellulose, or absorbable collagen) was sequentially applied to individual wounds and digitally compressed for 20 seconds. The wound was observed without pressure for 2 minutes. Up to 8 wounds per animal were created in 7 animals. For the swine splenic capsular stripping model a 2 x 2 cm area of capsular stripping on the surface of the spleen to a depth of 3 mm was created. Either p-GlcNAc membrane or oxidized cellulose was applied and digitally compressed for 60 seconds, followed by observation without pressure for 2 minutes. Six wounds per animal were created in 2 animals. If bleeding persisted in either model, a new cycle of compression was applied. These steps were repeated until hemostasis was achieved. No change in hemodynamics or coagulation factors was observed in either model. Subsequently, 10 consecutive patients undergoing elective small-bowel surgery were enrolled on pilot study. A 5 x 3 x 3 mm cruciate incision was created midway between the mesenteric and antimesenteric borders of the small bowel. Either p-GlcNAc membrane formulation or oxidized cellulose was applied (the sequence alternated per patient) with a 400-mg weight used for even, direct pressure. A second cruciate incision was then created on the contralateral side of the bowel to evaluate the second material. The number of applications required for hemostasis was assessed. Hemodynamics, small-bowel pathologic condition, and hematologic parameters were evaluated. RESULTS The p-GlcNAc membrane required fewer cycles of compression in the swine splenic incision model to achieve hemostasis than either absorbable collagen or oxidized cellulose (1.25 vs 2.58 and 3.41, respectively; P < .01) and caused more effective immediate cessation of bleeding (79% for p-GlcNAc vs 17% for both absorbable collagen and oxidized cellulose). With the more traumatic splenic capsular stripping model, p-GlcNAc required fewer cycles of compression to achieve hemostasis than oxidized cellulose (average, 2.5 versus 6.8 respectively; P < .01) and was able to achieve hemostasis with greater efficacy (50%) in 2 applications than did oxidized cellulose (0%; P < .01). When used in the human pilot study, p-GlcNAc membranes required fewer cycles of compression than oxidized cellulose (2.5 vs 5.4, respectively; P < .002), was able to stop bleeding with greater efficacy in 1 cycle of compression (50% vs 0%, respectively; P < .01), and ultimately accomplished hemostasis in 80% of the cases as opposed to 20%. CONCLUSIONS On the basis of its greater hemostatic efficacy as compared with collagen or oxidized cellulose-based products, p-GlcNAc holds promise as an effective topical hemostatic agent and deserves further evaluation.

Poly-N-Acetyl Glucosamine Nanofibers: A New Bioactive Material to Enhance Diabetic Wound Healing by Cell Migration and Angiogenesis

Introduction:In several fields of surgery, the treatment of complicated tissue defects is an unsolved clinical problem. In particular, the use of tissue scaffolds has been limited by poor revascularization and integration. In this study, we developed a polymer, poly-N-acetyl-glucosamine (sNAG), with bioactive properties that may be useful to overcome these limitations. Objective:To develop a scaffold-like membrane with bioactive properties and test the biologic effects in vitro and in vivo in diabetic wound healing. Methods:In vitro, cells–nanofibers interactions were tested by cell metabolism and migration assays. In vivo, full thickness wounds in diabetic mice (n = 15 per group) were treated either with sNAG scaffolds, with a cellulosic control material, or were left untreated. Wound healing kinetics, including wound reepithelialization and wound contraction as well as microscopic metrics such as tissue growth, cell proliferation (Ki67), angiogenesis (PECAM-1), cell migration (MAP-Kinase), and keratinocyte migration (p 63) were monitored over a period of 28 days. Messenger RNA levels related to migration (uPAR), angiogenesis (VEGF), inflammatory response (IL-1β), and extracellular matrix remodeling (MMP3 and 9) were measured in wound tissues. Results:sNAG fibers stimulated cell metabolism and the in vitro migratory activity of endothelial cells and fibroblasts. sNAG membranes profoundly accelerated wound closure mainly by reepithelialization and increased keratinocyte migration (7.5-fold), granulation tissue formation (2.8-fold), cell proliferation (4-fold), and vascularization (2.7-fold) compared with control wounds. Expression of markers of angiogenesis (VEGF), cell migration (uPAR) and ECM remodeling (MMP3, MMP9) were up-regulated in sNAG treated wounds compared with controls. Conclusions:The key mechanism of the bioactive membranes is the cell-nanofiber stimulatory interaction. Engineering of bioactive materials may represent the clinical solution for a number of complex tissue defects.

Effects of poly-N-acetyl glucosamine (pGlcNAc) patch on wound healing in db/db mouse.

BACKGROUND Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-based materials, produced by a marine microalga, have been characterized as effective hemostatic agents. In this study, we hypothesized that a pGlcNAc fiber patch may enhance wound healing in the db/db mouse. METHODS pGlcNAc patches were applied on 1-cm, full-thickness, skin wounds in the db/db mouse model. Wounds (n = 15 per group) were dressed with a pGlcNAc nanofiber patch for 1 hour, 24 hours, or left untreated. After the application time, patches were removed and wounds were allowed to heal spontaneously. The rate of wound closure was evaluated by digital analysis of unclosed wound area as a function of time. At day 10, wounds (n = 7 per group) were harvested and quantified with immunohistochemical markers of proliferation (Ki-67) and vascularization (platelet endothelial cell adhesion molecule). RESULTS Wounds dressed with pGlcNAc patches for 1 hour closed faster than control wounds, reaching 90% closure in 16.6 days, 9 days faster than untreated wounds. Granulation tissue showed higher levels of proliferation and vascularization after 1-hour treatment than the 24-hour and left-untreated groups. Foreign body reaction to the material was not noted in applications up to 24 hours. DISCUSSION In addition to its hemostatic properties, the pGlcNAc material also appears to accelerate wound closure in healing-impaired genetically diabetic mice. This material, with its combination of hemostatic and wound healing properties, has the potential to be effective agent for the treatment of complicated wounds.

Endoscopic injection of bleeding esophageal varices with a poly-N-acetyl glucosamine gel formulation in the canine portal hypertension model

BACKGROUND It has been shown that poly-N-acetyl glucosamine produces rapid hemostasis by stimulating erythrocyte aggregation. Endoscopic injection of this substance may be effective in the treatment of bleeding varices. METHODS In eight heparinized dogs with a bleeding esophageal varix greater than 2 mm in diameter, 2.5% to 3.5% poly-N-acetyl glucosamine gel was injected intravariceally and paravariceally. Endoscopy, endosonography, and histopathology were performed at 1, 7, 21, and 90 days after injection. RESULTS In all cases, the variceal hemorrhage was stopped with three to four injections of a mean total gel volume of 1.9 mL. No recurrence of bleeding, ulceration, or stricture formation occurred. Through replacement of the gel by connective tissue, the varix was permanently obliterated in its whole course in five cases and in more than 70% of its length in three cases. No embolization and no poly-N-acetyl glucosamine antibodies were detected. CONCLUSIONS Endoscopic injection of bleeding esophageal varices in this animal model with the use of poly-N-acetyl glucosamine gel was an effective and safe method for stopping the hemorrhage and inducing permanent varix obliteration.

Paracrine Release of IL-12 Stimulates IFN-γ Production and Dramatically Enhances the Antigen-Specific T Cell Response after Vaccination with a Novel Peptide-Based Cancer Vaccine

Interleukin-12 can act as a potent adjuvant for T cell vaccines, but its clinical use is limited by toxicity. Paracrine administration of IL-12 could significantly enhance the response to such vaccines without the toxicity associated with systemic administration. We have developed a novel vaccine delivery system (designated F2 gel matrix) composed of poly-N-acetyl glucosamine that has the dual properties of a sustained-release delivery system and a potent adjuvant. To test the efficacy of paracrine IL-12, we incorporated this cytokine into F2 gel matrix and monitored the response of OT-1 T cells in an adoptive transfer model. Recipient mice were vaccinated with F2 gel/SIINFEKL, F2 gel/SIINFEKL/IL-12 (paracrine IL-12), or F2 gel/SIINFEKL plus systemic IL-12 (systemic IL-12). Systemic levels of IL-12 were lower in paracrine IL-12-treated mice, suggesting that paracrine administration of IL-12 may be associated with less toxicity. However, paracrine administration of IL-12 was associated with an enhanced Ag-specific T cell proliferative and functional response. Furthermore, paracrine IL-12 promoted the generation of a stable, functional memory T cell population and was associated with protection from tumor challenge. To study the mechanisms underlying this enhanced response, wild-type and gene-deficient mice were used. The enhanced immune response was significantly reduced in IFN-γ−/− and IL-12Rβ2−/− recipient mice suggesting that the role of IL-12 is mediated, at least in part, by host cells. Collectively, the results support the potential of F2 gel matrix as a vaccine delivery system and suggest that sustained paracrine release of IL-12 has potential clinical application.

Isolation, purification, and characterization of poly-N-acetyl glucosamine use as a hemostatic agent.

BACKGROUND A new polymeric material, poly-N-acetyl glucosamine (p-GlcNAc) fiber, has been identified and is effective in achieving hemostasis in surgical procedures and trauma. The p-GlcNAc material is purified from large-scale cultures of a marine microalga. METHODS Poly-N-acetyl glucosamine materials have been formulated as films, sponges, gels, and microspheres. The polymers structure has been characterized by chemical composition, carbohydrate analysis, spectroscopic techniques, intrinsic viscosity, and electron microscopy. RESULTS Carbohydrate analyses indicate that the primary sugar present in p-GlcNAc is N-acetyl glucosamine. Elemental analyses yield percentage values for carbon, nitrogen, and hydrogen that support that the polymer is fully acetylated. Molecular weight determinations indicate that the polymer has a molecular weight of 2.0 x 10(6) Da. Fourier transform infrared, nuclear magnetic resonance, and circular dichroism spectral data have defined a unique tertiary structure. Biologic testing demonstrated that p-GlcNAc materials are fully biocompatible. CONCLUSION The p-GlcNAc fiber has a unique beta-tertiary structure.

Poly-N-acetyl glucosamine nanofibers regulate endothelial cell movement and angiogenesis: dependency on integrin activation of Ets1.

Poly-N-acetyl glucosamine (pGlcNAc) nanofiber-derived materials effectively achieve hemostasis during surgical procedures. Treatment of cutaneous wounds with pGlcNAc in a diabetic mouse animal model causes marked increases in cell proliferation and angiogenesis. We sought to understand the effect of the pGlcNAc fibers on primary endothelial cells (EC) in culture and found that pGlcNAc induces EC motility. Cell motility induced by pGlcNAc fibers is blocked by antibodies directed against αVβ3 and α5β1 integrins, both known to play important roles in the regulation of EC motility, in vitroand in vivo. pGlcNAc treatment activates mitogen-activated protein kinase and increases Ets1, vascular endothelial growth factor (VEGF) and interleukin 1 (IL-1) expression. pGlcNAc activity is not secondary to its induction of VEGF; inhibition of the VEGF receptor does not inhibit the pGlcNAc-induced expression of Ets1 nor does pGlcNAc cause the activation of VEGF receptor. Both dominant negative and RNA interference inhibition of Ets1 blocks pGlcNAc-induced EC motility. Antibody blockade of integrin results in the inhibition of pGlcNAc-induced Ets1 expression. These findings support the hypothesis that pGlcNAc fibers induce integrin activation which results in the regulation of EC motility and thus in angiogenesis via a pathway dependent on the Ets1 transcription factor and demonstrate that Ets1 is a downstream mediator of integrin activation.

Non-classical processes in surface hemostasis: mechanisms for the poly-N-acetyl glucosamine-induced alteration of red blood cell morphology and surface prothrombogenicity

It is well established that platelets and the intrinsic plasma coagulation pathway can be activated when blood contacts artificial surfaces. Experiments were performed to assess the effect of hemostatic poly-N-acetyl glucosamine (pGlcNAc) nanofibers on red blood cells. The pGlcNAc nanofibers, isolated from a marine diatom, interact with red blood cells (RBCs) to produce stomatocytes. The stomatocytes could be converted to echinocytes by treatment with echinocytic reagents, as measured by electron microscopy. Electrophoretic and Western blot analysis of RBC surface proteins demonstrated that pGlcNAc fibers were bound to band 3 of the RBC. An important and unique result of the interaction of RBCs with pGlcNAc fibers was the activation of the intrinsic coagulation cascade. This prothrombotic effect was associated with the presentation of phosphatidylserine on the outer layer of the surface membrane of nanofiber bound RBCs. The results demonstrate that RBCs can play a direct and important role in achieving surface hemostasis by accelerating the generation of thrombin, and add to the growing body of evidence that RBCs can strongly interact with hemostatic systems.

Breast cancer genome anatomy: correlation of morphological changes in breast carcinomas with expression of the novel gene product Di12.

To determine which genes may be activated or inactivated during breast cancer development, we employed two cloning strategies (subtractive hybridization and differential display) using RNA samples from a human breast tumor and its matching normal breast cell line. Of 950 clones isolated, 102 cDNA inserts were analysed by DNA sequencing and database searching. We found 30 clones that were obviously unidentified, with no significant homology to any listed human gene. We focused upon one of the novel genes, Di12, that is differentially expressed as a 1.35 kb RNA in breast cancer tissues and cell-lines, and in several normal tissues. A full length cDNA of this gene was cloned, and its DNA sequence revealed an open reading frame of 339 amino acids. Antibodies to the ten N-terminal amino acids were developed to investigate the expression of Di12 in breast cancer cell-lines and tumors. The Di12 protein was found in tissue sections of infiltrating ductal carcinomas (IDCs), but not in benign or normal breast specimens. RT – PCR analysis confirmed expression of Di12 in 80% of infiltrating ductal carcinomas (IDCs). As IDC constitutes ∼70% of breast cancers seen clinically, the level of Di12 expression may be predictive of disease progression.