Sustained release of granulocyte-macrophage colony-stimulating factor from a modular peptide-based cancer vaccine alters vaccine microenvironment and enhances the antigen-specific T-cell response.

The recent identification and molecular characterization of tumor antigens provides the opportunity to explore the rational development of peptide-based cancer vaccines. However, the response to these vaccines remains variable, and peptide-based cancer vaccines may even produce tolerance induction and enhanced tumor growth. The authors have developed a unique method for the isolation of a polysaccharide polymer of chemically pure poly- N -acetyl glucosamine (p-GlcNAc). This highly purified polysaccharide can be formulated into a stable gel matrix (designated F2 gel matrix) with unique properties of a sustained-release delivery system that has previously been shown to be an effective immune adjuvant. F2 gel matrix is capable of providing sustained release of antigenic peptide and cytokine in vitro. The purposes of this study were to characterize the ability of F2 gel matrix to provide sustained local release of cytokines in vivo and to test the hypothesis that such sustained release can enhance the microenvironment for antigen presentation, leading to a more effective antitumor response. Subcutaneous administration of F2 gel/cytokine matrix resulted in sustained release of cytokine at the vaccine site for up to 120 hours. Sustained release of granulocyte-macrophage colony-stimulating factor (GM-CSF) was associated with an increased inflammatory infiltrate at the vaccine site and enhanced dendritic cell activation. Further, accination with F2 gel/SIINFEKL/GM-CSF matrix resulted in enhanced antigen-specific immunity. Addition of GM-CSF to the F2 gel matrix resulted in an increase in the percentage of antigen-specific T cells in the draining lymph nodes, enhanced cytotoxicity, a sustained presence of antigen-specific T cells in the peripheral blood, and protection from E.G7 tumor challenge. These results support the potential of an F2 gel matrix modular vaccine delivery system that can provide sustained local release of cytokine in vivo, and confirm the powerful effects of GM-CSF as an immune adjuvant.

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.

Primary T-cell and activated macrophage response associated with tumor protection using peptide/poly-N-acetyl glucosamine vaccination.

The mode of peptide-based cancer vaccine administration critically affects the ability to achieve a clinically relevant tumor-specific response. We have previously shown (Cole et al., Clin. Cancer Res., 3: 867-873, 1997) that a specific formulation of the polysaccharide poly-N-acetyl glucosamine (p-GlcNAc, designated as F2 gel) is an effective vehicle for sustained cytokine and peptide delivery in vitro. The purpose of this study was to evaluate the efficacy of F2 gel/peptide vaccination in the murine EG.7-OVA tumor model and to elucidate potential mechanisms involved in the observed cell-mediated response. C57BL/6 mice were given injections of 200 microl in the base of tail/footpad using either F2 gel alone or 200 microg of: SIINFEKL minimal peptide (OVA) in PBS, OVA peptide/endoplasmic reticulum insertion signal sequence fusion (ESOVA) in PBS, OVA in F2 gel, or ESOVA in F2 gel. Splenocytes were tested 10 days later for a secondary response using a Cr51 assay as well as a primary CTL response using the lactate dehydrogenase cytotoxicity assay. Splenocytes from immunized mice were harvested at specific time points and assayed for cell surface and intracellular markers. On day 10 postvaccination, animals were challenged with EG.7-OVA murine thymoma cells. Tumor size and appearance were recorded. Vaccination with F2 gel/peptide (either OVA or ESOVA) resulted in a primary T-cell response (up to 25% tumor cell-specific lysis) and no tumor growth in 69% of the mice. By 48 h, the proportion of splenic T cells had increased 4-fold compared with B cells. Presence of an increased Th1 CD4 helper population was demonstrated by IFN-gamma production. CD4 cells were activated at 24 and 48 h as shown by IL-2 receptor alpha chain expression (from 2% basal expression to 15.4% at 48 h). Activated splenic macrophages increased from 3 to 8% within 10 h, and their level of B7-2 expression doubled. Depletion of macrophages before vaccine injection abolished any tumor-specific primary CTL response. F2 gel/peptide tumor vaccine can prime the immune system in an antigen-specific manner by generating a measurable primary T-cell response with minimal peptide; this process involves macrophage presence and activation as well as induction of Th1 CD4 cells. This is the first demonstration of a primary CTL response generated with minimal peptide vaccination using a noninfectious delivery system. These results justify additional studies to better define the mechanisms involved in F2 gel/peptide vaccination in preparation for clinical trials.

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.

Monoclonal antibodies against metalloporphyrins. Specificity of interaction with structurally different metalloporphyrins.

Monoclonal antibodies against Pd-coproporphyrin I have been obtained. The antibody specificity for free as well as for conjugated Pd-coproporphyrin I is characterized. Affinity constants are estimated for 3 monoclonal antibodies effectively interacting with free Pd-coproporphyrin I. A comparative study on the binding of monoclonal antibodies with analogues and derivatives of Pd-coproporphyrin I has revealed that the antigen is mainly located inside the antibody paratope. The protein adjoins complementary to the metalloporphyrin in such a manner that antibodies obtained discern only isomer I, and to some degree, isomer III of coproporphyrin.

[Co-oxidation of phenols and 4-aminoantipyrene catalyzed by microperoxidase and their complexes with proteins]. / Sopriazhennoe okislenie fenolov i 4-aminoantipirina, kataliziruemoe mikroperoksidazami i ikh kompleksami s belkami.

The kinetic parameters of 4-aminoantipyrine (AAP) co-oxidation with phenol or p-iodophenol in a wide range of hydrogen peroxide concentrations were compared for microperoxidases MP-8, MP-9, MP-11, horseradish peroxidase (HRP) and peroxidase of Arthromyces ramosus (PAR). The peroxidatic activity increased in the following order: HRP > PAR > MP-9 > MP-8 > MP-11. Microperoxidase complexes with human serum albumin (1:1) retained their peroxidatic activity at high H2O2 concentrations, protecting haem from destruction by active radicals. Monoclonal antibodies against porphyrin decreased the peroxidatic activities of three microperoxidases during co-oxidation of AAP and phenol and protected haem. Inert proteins (BSA, ovalbumin) had little effect on the HRP activity, whereas enzyme-specific antibodies strongly activated HRP at high concentrations of hydrogen peroxide, thereby forming an immune complex protecting HRP from its dissociation down to haem and the apoenzyme.

Universal phosphorescence immunoassay.

The aim of this study is to develop a universal phosphorescence immunoassay method using monoclonal antibodies to Pd-coproporphyrin (Pd-CP) and conjugates of various proteins with Pd-CP. Pd-CP and monoclonal antibodies obtained allow a convenient method for the determination of various antigens to be developed. The conditions for immunological reactions with Pd-CP were optimized with respect to the components affecting the nonspecific binding Pd-CP and Pd-CP conjugates.

Kinetics of oxidation of o-dianisidine by hydrogen peroxide in the presence of antibody complexes of iron(III) coproporphyrin.

The complex of iron(III) coproporphyrinI (FeCPI) with antibody D5E3 was studied as an artificial peroxidase, using o-dianisidine as a substrate. At saturation with respect to antibody, the initial rates of o-dianisidine oxidation are practically the same for free and bound FeCPI at a concentration 5 x 10(-9)M, but the catalytic rate constant (kc) for bound FeCPI exceed (kc) for free FeCPI by two- to three-fold. This difference can be explained by a real enhancement of (kc) at the antibody-active site. The dependence of initial rates of the reaction on substrate concentrations obeyed Michaelis-Menten kinetics and revealed substrate activation at high concentrations of o-dianisidine. A comparison of the Stern-Volmer constants for o-dianisidine-induced quenching of the porphyrin fluorescence proves that antibody-bound coproporphyrin is equivalently accessible to the substrate as protoporphyrin bound to apoperoxidase from horseradish peroxidase (HRP). Based on analysis of the (kc) dependence on H2O2 concentrations in the FeCPI-antibody system, we suggest that interaction with hydrogen peroxide is the rate-limiting step for the oxidation reaction.

[Formation of a pentose phosphate cycle metabolite, erythrose-4-phosphate, from initial compounds of glycolysis by transketolase from the rat liver]. / Obrazovanie metabolita pentozofosfatnogo puti--éritrozo-4-fosfata--iz nachal'nykh soedinenii glikoliza transketolazoi iz pecheni krysy.

Using ion-exchange chromatography of sucrose phosphates on Dowex-1, it was demonstrated that the highly purified rat liver transketolase (specific activity 1.7 mumol/min.mg protein) is capable of catalyzing the synthesis of erythrose-4-phosphate, a metabolite of the pentose phosphate pathway non-oxidizing step, from the initial participants of glycolysis, i. e., glucose-6-phosphate and fructose-6-phosphate. As can be evidenced from the reaction course, the second product of this synthesis is octulose-8-phosphate. The reaction was assayed by accumulation of erythrose-4-phosphate. The soluble fraction from rat liver catalyzes under identical conditions the synthesis of heptulose-7-phosphate (but not erythrose-4-phosphate), which points to the utilization of the erythrose-4-phosphate formed in the course of the transketolase reaction by transaldolase which is also present in the soluble fraction. The role of the transketolase reaction reversal from the synthesis of pentose phosphate derivatives to glycolytic products is discussed. The transketolase reaction provides for the relationship between glycolysis and the anaerobic step of the pentose phosphate pathway which share common metabolites, i. e. glucose-6-phosphate and fructose-6-phosphate.