Association between wetland presence and incidence of Salmonella enterica serotype Javiana infections in selected US sites, 2005-2011.

Salmonella causes an estimated 1·2 million illnesses annually in the USA. Salmonella enterica serotype Javiana (serotype Javiana) is the fourth most common serotype isolated from humans, with the majority of illnesses occurring in southeastern states. The percentage of wetland cover by wetland type and the average incidence rates of serotype Javiana infection in selected counties of the Foodborne Disease Active Surveillance Network (FoodNet) were examined. This analysis explored the relationship between wetland environments and incidence in order to assess whether regional differences in environmental habitats may be associated with observed variations in incidence. Findings suggest that environmental habitats may support reservoirs or contribute to the persistence of serotype Javiana, and may frequently contribute to the transmission of infection compared with other Salmonella serotypes.

The rise and decline in Salmonella enterica serovar Enteritidis outbreaks attributed to egg-containing foods in the United States, 1973-2009.

Salmonella enterica causes an estimated 1 million domestically acquired foodborne illnesses annually. Salmonella enterica serovar Enteritidis (SE) is among the top three serovars of reported cases of Salmonella. We examined trends in SE foodborne outbreaks from 1973 to 2009 using Joinpoint and Poisson regression. The annual number of SE outbreaks increased sharply in the 1970s and 1980s but declined significantly after 1990. Over the study period, SE outbreaks were most frequently attributed to foods containing eggs. The average rate of SE outbreaks attributed to egg-containing foods reported by states began to decline significantly after 1990, and the proportion of SE outbreaks attributed to egg-containing foods began declining after 1997. Our results suggest that interventions initiated in the 1990s to decrease SE contamination of shell eggs may have been integral to preventing SE outbreaks.

Attribution of Salmonella enterica serotype Hadar infections using antimicrobial resistance data from two points in the food supply system.

A challenge to the development of foodborne illness prevention measures is determining the sources of enteric illness. Microbial subtyping source-attribution models attribute illnesses to various sources, requiring data characterizing bacterial isolate subtypes collected from human and food sources. We evaluated the use of antimicrobial resistance data on isolates of Salmonella enterica serotype Hadar, collected from ill humans, food animals, and from retail meats, in two microbial subtyping attribution models. We also compared model results when either antimicrobial resistance or pulsed-field gel electrophoresis (PFGE) patterns were used to subtype isolates. Depending on the subtyping model used, 68-96% of the human infections were attributed to meat and poultry food products. All models yielded similar outcomes, with 86% [95% confidence interval (CI) 80-91] to 91% (95% CI 88-96) of the attributable infections attributed to turkey, and 6% (95% CI 2-10) to 14% (95% CI 8-20) to chicken. Few illnesses (<3%) were attributed to cattle or swine. Results were similar whether the isolates were obtained from food animals during processing or from retail meat products. Our results support the view that microbial subtyping models are a flexible and robust approach for attributing Salmonella Hadar.

Quantitative exposure assessment of waterfowl hunters to avian influenza viruses.

The potential for direct transmission of type A influenza viruses from wild waterfowl to humans is undefined. This study estimated exposure of hunters to avian influenza virus (AIV) resulting from direct contact with potentially infected waterfowl in Georgia (GA), Louisiana (LA) and Minnesota (MN), and demonstrated variation in the risk of exposure to AIV by hunting location and time. Hunting begins earlier in MN, starting in October, and later in GA and LA, usually starting in November. In addition, the numbers of hunters and birds harvested varies considerably in each state, with LA hosting the largest harvest in the USA Temporal effects resulted in variation of the exposure risk per hunter-day, with a higher risk associated with the earlier months of the hunting season. Exposure risk in locations varied due to AIV prevalence during each hunting season, average bird harvest per hunter-day, and ratio of juveniles/adult birds harvested (higher risk associated with higher ratios). Population risk is discussed based on the exposure risk and number of active hunters in each state per month. The risk of human exposure to AIV was also shown to be temporally distinct from the time of greatest risk of human influenza A infection during circulation of seasonal human influenza viruses, making recombination events due to co-infection unlikely.

Foodborne outbreaks of shigellosis in the USA, 1998-2008.

We examined reported outbreaks of foodborne shigellosis in the USA from 1998 to 2008 and summarized demographic and epidemiological characteristics of 120 confirmed outbreaks resulting in 6208 illnesses. Most reported foodborne shigellosis outbreaks (n = 70, 58%) and outbreak-associated illnesses (n = 3383, 54%) were restaurant-associated. The largest outbreaks were associated with commercially prepared foods distributed in multiple states and foods prepared in institutional settings. Foods commonly consumed raw were implicated in 29 (24%) outbreaks and infected food handlers in 28 (23%) outbreaks. Most outbreaks (n = 86, 72%) were caused by Shigella sonnei. Targeted efforts to reduce contamination during food handling at multiple points in the food processing and distribution system, including food preparation in restaurants and institutional settings, could prevent many foodborne disease outbreaks and outbreak-related illnesses including those due to Shigella.

Are Surgical Drains Needed in DIEP Flap Surgery? The Drain-Free DIEP Flap Concept.

BACKGROUND: Studies support an inherent morbidity associated with the use of surgical drains-such as postoperative pain, infection, reduction in mobility, and delay in patient discharge-and they do not prevent seroma or hematoma. The authors' series aims to evaluate the feasibility, benefits, and safety of performing drainless deep inferior epigastric perforator (DIEP) flap surgery and to formulate an algorithm for when this can be used. METHODS: A retrospective review of DIEP reconstruction outcomes of two surgeons was performed. Over the course of 24 months, consecutive DIEP flap patients were included from the Royal Marsden Hospital in London and Austin Hospital in Melbourne, and drain use, drain output, length of stay (LOS), and complications were analyzed. RESULTS: A total of 107 DIEP flap reconstructions were performed by two surgeons. Thirty-five patients had abdominal drainless DIEP flaps, and 12 patients had totally drainless DIEP flaps. Mean age was 52 years (range, 34 to 73 years) and mean body mass index was 26.8 kg/m 2 (range, 19.0 to 41.3 kg/m 2 ). Abdominal drainless patients showed a potential trend toward shorter hospital stays as compared with the ones with drains (mean LOS, 3.74 days versus 4.05 days; P = 0.154). Totally drainless patients had an even shorter, statistically significant, mean LOS of 3.10 days, as compared with patients with drains (4.05 days, P = 0.002), with no increase in complications. CONCLUSIONS: The avoidance of abdominal drains in DIEP flaps reduces hospital stay without increasing complications, and this has become our standard practice for patients with a body mass index of less than 30 kg/m 2 . It is our opinion that the totally drainless DIEP flap procedure is safe in selected patients. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, III.

Carotid artery disease: stenting vs endarterectomy.

Several multicentre, randomized trials have validated the efficacy of carotid endarterectomy (CEA). Comparative randomized trials are also currently developing insight into the role of carotid angioplasty and stenting (CAS), and identifying factors for optimal patient selection. Although these interventions are aimed at embolic stroke prevention, anaesthetic management might prevent the subset of strokes that are haemodynamic in nature by maintaining tight physiological control. The perioperative risk of myocardial events is increased in this population. Hence, preoperative attention to cardiovascular disease, hypertension, renal insufficiency, and diabetes mellitus might reduce neurological and cardiovascular complications. During carotid artery cross-clamping, the risk of cerebral ischaemia can be decreased by maintaining normal to high perfusion pressure. Although there is no demonstrable advantage of a specific anaesthetic technique for patients undergoing CEA, it is imperative that cerebral blood flow is optimized, that there is minimal cardiac stress, and that anaesthetic recovery is rapid. Carotid angioplasty and stenting is performed under light sedation with antithrombotic therapy and vigilance for bradycardia and hypotension. Tight haemodynamic control remains a priority in the immediate postoperative period for both interventions.

Survey of biosecurity protocols and practices adopted by growers on commercial poultry farms in Georgia, U. S. A.

The integrated commercial poultry system is a highly connected network in which routine activities keep farms within a geographic area in constant contact. Consequently, biosecurity practices designed to minimize the transmission of infectious diseases between and within farms are an important component of modern flock health programs. A survey of Georgia poultry growers was conducted in order to assess the level of adoption of standard biosecurity measures by farm personnel and visitors. The results showed that compliance with recommended biosecurity practices did not significantly vary by company, farm size, or number of farms owned by the same grower. However, biosecurity was higher in the northern part of the state, where the density of farms is higher, and where there was an ongoing outbreak of infectious laryngotracheitis at the time of the study. The survey found that growers place more emphasis on biosecurity measures targeting farm visitors than those targeting farm personnel. Most growers reported that all visitors to the farm were required to wear shoe covers, although visitors were not typically required to park outside the farm entrance or to wash tires on their vehicles. No visitor type was reportedly excluded from poultry houses during grow out on all farms. The results highlight the need to evaluate the comparative efficacy of specific biosecurity measures in order to set priorities and attain feasible rates of implementation of targeted biosecurity practices.

Stochastic model of the potential spread of highly pathogenic avian influenza from an infected commercial broiler operation in Georgia.

The potential spread of highly pathogenic avian influenza among commercial broiler farms in Georgia, U. S. A., was mathematically modeled. The dynamics of the spread within the first infected flock were estimated using an SEIR (susceptible-exposed-infectious-recovered) deterministic model, and predicted that grower detection of flock infection is most likely 5 days after virus introduction. Off-farm spread of virus was estimated stochastically for this period, predicting a mean range of exposed farms from 0-5, depending on the density of farms in the area. Modeled off-farm spread was most frequently associated with feed trucks (highest daily probability and number of farm visits) and with company personnel or hired help (highest level of bird contact).

Decreased T lymphocyte migration in patients with malignancy mediated by a suppressor cell population.

The migration and concentration of lymphocytes at sites of antigenic challenge are an integral part of the expression of delayed cutaneous hypersensitivity, as well as of tumor and graft rejection. In this study, we have analyzed the migration of T lymphocytes from patients with malignancy. We used casein and concanavalin A (Con A)-stimulated mononuclear cell supernatants to stimulate T cell locomotion. Peripheral blood T lymphocytes from 30 patients with established malignancy, 10 patients with indolent malignancy or benign tumor, and 42 normal adult controls were tested. Data are expressed as a migration index (MI), which represents the difference in micrometers between the distance migrated in response to a stimulus and the distance migrated in response to media alone. We observed a marked depression in casein-stimulated T lymphocyte migration in patients with established malignancy (mean MI +/- 1 SD = 17.0 +/- 9 microns) as compared with normal adult controls (mean MI +/- 1 SD = 35.3 +/- 10 microns). Similar results were observed with migration in response to Con A supernatants. T cells from patients with established malignancy had a mean MI of 5.8 +/- 4 microns to Con A supernatants as compared with 24.5 +/- 5 for controls. This depressed migration was apparent both in the distance that cells migrated and in the number of cells that migrated into the membrane. Of 10 patients with indolent malignancy or benign tumor, T cell migration in 8 was not significantly decreased as compared with controls. When we mixed equal concentrations of normal control T lymphocytes with T lymphocytes from patients with cancer and added the mixture directly to the upper compartment of the chemotaxis chamber, the response of the normal T cells to casein was inhibited by an average of 48%. We observed inhibition of this migration of normal cells when we added as little as 10% of patient cells to normal cells. When we mixed normal control T lymphocytes from different donors and added them directly to the upper compartment of the chemotaxis chamber, T lymphocyte migration in response to casein was not significantly altered. If T cells from patients with cancer were cultured overnight, the suppressive effect on lymphocyte locomotion was lost. Our results indicate that there is a population of T lymphocytes in patients with cancer that suppress normal T lymphocyte migration. This suppressor activity may partially explain the subversion of immunosurveillance in established neoplastic states, as well as the defective inflammatory reaction to intradermal injection of antigen observed in many patients with malignancy.

Glutathione-mediated detoxification systems in plants.

Recent work has highlighted the presence of diverse glutathione-dependent enzymes in plants with potential roles in the detoxification of both xenobiotic and endogenous compounds. In particular, studies on glutathione transferases are further characterising their role in xenobiotic metabolism, and also raising intriguing possible roles in endogenous metabolism. The solution of their three-dimensional structures together with studies on their molecular diversity and substrate specificity is providing new insights into the function and classification of these enigmatic enzymes.

Phase I trial of recombinant human macrophage colony-stimulating factor administered by continuous intravenous infusion in patients with metastatic cancer.

BACKGROUND: Macrophage colony-stimulating factor is a bone marrow-derived glycoprotein that can stimulate monocytes and macrophages, resulting in production of factors involved in immune response. In vitro and in vivo preclinical studies in animals have demonstrated that recombinant human macrophage colony-stimulating factor (rHuM-CSF) can have antitumor activity. PURPOSE: A phase I clinical trial was undertaken to evaluate the toxicity, pharmacokinetics, and immunologic effects of rHuM-CSF given by continuous intravenous infusion in patients with cancer. METHODS: Eighteen patients with metastatic solid tumors refractory to conventional therapy were treated with rHuM-CSF. Twelve patients received two 14-day cycles of rHuM-CSF by continuous infusion, with a 2-week interval. Dose escalation levels were 50, 100, and 150 micrograms/kg over 24 hours. Consecutive cohorts of three to six patients were planned at each dose level. Six patients received a modified regimen of four 7-day periods of infusion at 100 micrograms/kg over 24 hours, with 1-week intervals. RESULTS: Dose-limiting toxicity was grade 4 thrombocytopenia at a dose of 150 micrograms/kg over 24 hours in two patients receiving the 2-week regimen. Platelet count nadirs and concomitant monocytosis were seen on days 7-9, but recovery occurred during the treatment period. Macrophage colony-stimulating factor serum levels were maximal on day 1 and returned to near baseline on day 7 of infusion. Patients treated with four 7-day infusions had no treatment-limiting thrombocytopenia. There were no cumulative effects on platelet or monocyte counts or significant constitutional symptoms. Subclinical conjunctival injection was noted in five of 10 patients receiving screening ophthalmologic evaluation. Grade 2 episcleritis was diagnosed in one patient, and asymptomatic perilimbal and retinal hemorrhages were seen in two. Two patients developed sepsis caused by the intravenous line, which required cessation of therapy. No objective responses were documented. CONCLUSION: The maximum tolerated dose of rHuM-CSF given by continuous intravenous infusion for 14 days was 100 micrograms/kg over 24 hours, with rapidly reversible, dose-limiting thrombocytopenia at 150 micrograms/kg over 24 hours. A regimen alternating weekly cycles of infusion avoids dose-limiting toxicity and allows long-term treatment. IMPLICATIONS: The regimen of repeated 7-day infusions may be useful for future studies evaluating rHuM-CSF-activated monocytes in therapy for long-term infectious diseases or in investigation of new modes of cancer therapy using rHuM-CSF in conjunction with a tumor-specific antibody.

T-cell receptor repertoire in matched MART-1 peptide-stimulated peripheral blood lymphocytes and tumor-infiltrating lymphocytes.

Characterization of tumor-associated antigens (TAAs) recognized by CTLs makes the consideration of therapeutic strategies based on peptide stimulation of peripheral blood lymphocytes (PBLs) feasible. Several such approaches are adoptive transfer of peptide-stimulated PBLs, ex vivo peptide stimulation of dendritic cells, and direct vaccination with TAA-derived peptides. A critical component of any of these peptide-based strategies is the requirement that the patient's PBLs are able to react productively against the presented TAA. The purpose of this study, through the study of T-cell receptor (TCR) usage, was to evaluate the T-cell response in matched MART-1(27-35) peptide-stimulated PBLs and tumor-infiltrating lymphocytes (TILs). MART-1(27-35)-reactive PBL and TIL cultures were generated from three patients by in vitro stimulation with an immunodominant peptide of MART-1 (MART-1(27-35)). All cultures had a human leukocyte antigen A2-restricted, MART-1(27-35)-specific CTL response. The TCR usage of each was assessed by the DNA sequence analysis of 50 TCR beta clones obtained by rapid amplification of cDNA ends per culture. TCR analysis suggests a TCR repertoire that differed from patient to patient (8-16 subfamilies were used) and a predominant usage of a different variable beta chain (BV) by each of these MART-reactive T cells. These predominant BV rearrangements were derived from multiple clonotypes because different variable, diversity, and junctional regions were observed. However, a similar pattern of expansion was present for both PBLs and TILs; the relative usage of each prevailing BV was more marked in TILs (36, 50, and 78% of TILs versus 26, 20, and 24% of PBLs, respectively), a broader TCR repertoire was used by PBLs (P > 0.05), and similar TCR subfamily usage was noted when TIL and PBL cultures from the same patient were compared (8 of 11, 7 of 9, and 7 of 8 for patients 1, 2, and 3, respectively). Furthermore, the exact same clonotypes derived from predominant TCR subfamilies in the PBLs and TILs were present in each patient, suggesting peptide-stimulated expansion in both biological compartments. These studies suggest that there will not be a limited and predictable TCR subfamily response to a specific TAA, although reproducible patterns of PBL and TIL expansion are present from patient to patient. Additionally, identical T-cell clonotypes having the same potential for antigen-driven expansion were present in a patient's PBLs and TILs. As such, our data support the conceptualization of approaches using adoptive transfer or vaccination based on TAA-derived peptide stimulation of PBLs.

Characterization of the functional specificity of a cloned T-cell receptor heterodimer recognizing the MART-1 melanoma antigen.

T cells can play a central role in the immune response to cancer, with tumor-specific T-lymphocyte reactivity provided by the T-cell receptor (TCR) alpha and beta chain heterodimer. This study is the first report of the definitive identification and characterization of a functional tumor-associated, antigen-specific TCR by reconstitution in an alternate cell line. Jurkat T cells were transfected with the cDNAs encoding the full-length alpha and beta T-cell receptor chains from the HLA-A2 restricted, melanoma-reactive T-cell clone, clone 5. Expression of the transfected TCR was evaluated by immunofluorescence after down-modulation of the endogenous receptor with Jurkat T-cell receptor beta chain-specific mAb. Jurkat clone 5 TCR+ cells recognized MART-1 peptides presented by T2 cells in a pattern and sensitivity equivalent to native MART-1-reactive T-cells. Recognition of HLA-A2+ melanoma cell lines by the Jurkat clone 5 TCR+ cells, however, did not occur without the addition of exogenous MART-1 peptide. The cloning and expression of functional TCR genes which are capable of specifically recognizing MART-1 antigen provides reagents which could be used for the study of the mechanisms of T-cell/tumor antigen interactions and creates immortalized reagents which can facilitate studies requiring detection of the MART-1 antigen. The tumor reactivity provided by these genes could also have application in novel immunotherapeutic strategies for treating patients with melanoma, including redirection of tumor-infiltrating lymphocyte specificity and bone marrow stem cell therapy.

Identification of MART-1-specific T-cell receptors: T cells utilizing distinct T-cell receptor variable and joining regions recognize the same tumor epitope.

Tumor-specific cytotoxic T lymphocytes (CTLs) can mediate tumor regression in patients with metastatic melanoma and play a central role in the immune response to cancer. The recent identification of shared melanoma antigens has raised the possibility of a limited melanoma-specific T-cell receptor (TCR) repertoire, but subsequent studies have been controversial and difficult to interpret without knowing which tumor-associated antigens (TAAs) are being recognized by specific TCRs. However, the recent cloning of several melanoma TAAs now allows for the identification of the specifically recognized TAA and its epitope. We evaluated the TCR of two clonal CD8+ CTL lines, A42 and 1E2, from two HLA-A2+ patients with metastatic melanoma. Both CTL lines were MART-1 specific, and both demonstrate reactivity to the same epitope when presented in an HLA-A2.1 context. The TCR genes of the two clones were sequenced. All of the productively rearranged A42 TCR beta chain genes were V beta 7/D beta 2.1/J beta 2.7/C beta 2; the TCR alpha chain genes were V alpha 21/J alpha 42/C alpha. The 1E2 TCR beta chain genes were V beta 3/D beta 1.1/J beta 1.1/C beta 1, and TCR alpha chains were V alpha 25/J alpha 54/C alpha. This study is the first report of TCR sequences specific for a melanoma epitope. These TCR clones may be useful for the development of more effective immunotherapies and in studies of the mechanism of T-cell recognition of tumor antigen. They also provide direct evidence that the immune system can provide more than one TCR capable of recognizing a TAA epitope.

The CaSm (LSm1) oncogene promotes transformation, chemoresistance and metastasis of pancreatic cancer cells.

The cancer-associated Sm-like (CaSm) oncogene is overexpressed in 87% of human pancreatic tumor samples and CaSm knockdown has demonstrated therapeutic efficacy in murine models of pancreatic cancer. Evidence indicates that CaSm modulates messenger RNA degradation; however, its target genes and the mechanisms by which CaSm promotes pancreatic cancer remain largely unknown. Here, we demonstrate that the CaSm overexpression alters several hallmarks of cancer-including transformation, proliferation, chemoresistance and metastasis. Doxycycline-induced CaSm expression enhanced proliferation and both anchorage-dependent and -independent growth of the human Panc-1 cells in vitro. CaSm induction decreased gemcitabine-induced cytotoxicity and altered the expression of apoptotic regulation genes, including Bad, E2F1 and Bcl-XL. CaSm-overexpressing Panc-1 cells were twofold more migratory and fourfold more invasive than the driver controls and demonstrated characteristics of epithelial-to-mesenchymal transition such as morphological changes and decreased E-cadherin expression. CaSm induction resulted in changes in RNA expression of metastasis-associated genes such as MMP1, SerpinB5, uPAR and Slug. Using a murine model of metastatic pancreatic cancer, injection of CaSm-induced Panc-1 cells resulted in a higher abundance of hepatic metastatic lesions. Overall, CaSm overexpression contributed to a more aggressive cancer phenotype in Panc-1 cells, further supporting the use of CaSm as a therapeutic target against pancreatic cancer.

Randomized comparison of high-dose and low-dose intravenous interleukin-2 for the therapy of metastatic renal cell carcinoma: an interim report.

PURPOSE: A randomized prospective study was performed to compare the efficacy and toxicity of high-dose intravenous bolus interleukin-2 (IL-2) and a lower-dose intravenous bolus regimen for the treatment of metastatic renal cell carcinoma (RCC). PATIENTS AND METHODS: Between March 1991 and April 1993, 125 patients with metastatic RCC were randomized to receive IL-2 by intravenous bolus every 8 hours at either 720,000 IU/kg (high-dose) or 72,000 IU/kg (low-dose) to the maximum-tolerated number of doses (or a maximum of 15 doses). After approximately 7 to 10 days, both treatment groups were re-treated with a second identical cycle of therapy. Those patients who were stable or responding to treatment 5 to 6 weeks later went on to receive re-treatment with another course (two cycles) of therapy. Response rates and toxicity were determined for the two treatment arms. RESULTS: One hundred twenty-five patients received a total of 208 courses of therapy. Sixty patients were randomized to receive low-dose, and 65 to receive high-dose IL-2. There were no treatment-related deaths in either arm. There was a greater incidence of grade III or IV thrombocytopenia, malaise, and hypotension in patients who received high-dose IL-2, while patients who received low-dose IL-2 had significantly more infections. Three percent of treatment courses with low-dose IL-2 required vasopressor support, compared with 52% of courses with high-dose IL-2. Patients who received low-dose IL-2 had a 7% complete response (CR) and an 8% partial response (PR) rate, and patients who received high-dose IL-2 had a 3% CR and a 17% PR rate. CONCLUSION: Low-dose intravenous bolus IL-2 represents an effective regimen for the treatment of metastatic RCC, with preliminary results comparable to those observed with high-dose IL-2. Low-dose IL-2 can be administered with significantly fewer complications, reduced use of vasopressor support, and fewer admissions to an intensive care unit (ICU).

The structure of a zeta class glutathione S-transferase from Arabidopsis thaliana: characterisation of a GST with novel active-site architecture and a putative role in tyrosine catabolism.

The cis-trans isomerisation of maleylacetoacetate to fumarylacetoacetate is the penultimate step in the tyrosine/phenylalanine catabolic pathway and has recently been shown to be catalysed by glutathione S-transferase enzymes belonging to the zeta class. Given this primary metabolic role it is unsurprising that zeta class glutathione S-transferases are well conserved over a considerable period of evolution, being found in vertebrates, plants, insects and fungi. The structure of this glutathione S-transferase, cloned from Arabidopsis thaliana, has been solved by single isomorphous replacement with anomalous scattering and refined to a final crystallographic R-factor of 19.6% using data from 25.0 A to 1.65 A. The zeta class enzyme adopts the canonical glutathione S-transferase fold and forms a homodimer with each subunit consisting of 221 residues. In agreement with structures of glutathione S-transferases from the theta and phi classes, a serine residue (Ser17) is present in the active site, at a position that would allow it to stabilise the thiolate anion of glutathione. Site-directed mutagenesis of this residue confirms its importance in catalysis. In addition, the role of a highly conserved cysteine residue (Cys19) present in the active site of the zeta class glutathione S-transferase enzymes is discussed.

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.

Characterization of a sustained-release delivery system for combined cytokine/peptide vaccination using a poly-N-acetyl glucosamine-based polymer matrix.

Identification of tumor-associated antigens (TAAs) and their class I MHC-restricted epitopes now allows for the rational design of peptide-based cancer vaccines. A biocompatible system capable of sustained release of biologically relevant levels of cytokine and TAA peptide could provide a more effective microenvironment for antigen presentation. Our goal was to test a sustained-release cytokine/TAA peptide-based formulation using a highly purified polysaccharide [poly-N-acetyl glucosamine (p-GlcNAc)] polymer. Granulocyte-macrophage colony-stimulating factor (GM-CSF; 100 microgram) and MART-1(27-35) peptide (128 microgram in DMSO) were formulated into p-GlcNAc. Peptide release was assayed in vitro using interleukin 2 production from previously characterized MART-1(27-35)-specific Jurkat T cells (JRT22). GM-CSF release was assayed via ELISA and proliferation of M-07e (GM-CSF-dependent) cells. Local bioavailability of MART-1(27-35) peptide for uptake and presentation by antigen-presenting cells was demonstrated for up to 6 days (>0.5 microgram/ml). More than 1.0 microgram/ml GM-CSF was concomitantly released over the same period. Biocompatibility and local tissue response to p-GlcNAc releasing murine GM-CSF was determined in C57BL/6 mice via s.c. injection using murine GM-CSF (0. 2 microgram/ml) in 200 microliter of a 2.5% polymer gel. Significant lymphocytic and eosinophilic infiltration was observed 2-7 days after injection with polymer containing murine GM-CSF. The results of our studies show that this biocompatible system is capable of a sustained concomitant release of biologically active peptide and cytokine into the local microenvironment. These findings support further studies to validate a p-GlcNAc delivery system vehicle for a cytokine/TAA peptide-based cancer vaccine.