Efficacy of a Vaccine Based on Protective Antigen and Killed Spores against Experimental Inhalational Anthrax

Protective antigen (PA)-based anthrax vaccines acting on toxins are less effective than live attenuated vaccines, suggesting that additional antigens may contribute to protective immunity. Several reports indicate that capsule or spore-associated antigens may enhance the protection afforded by PA. Addition of formaldehyde-inactivated spores (FIS) to PA (PA-FIS) elicits total protection against cutaneous anthrax. Nevertheless, vaccines that are effective against cutaneous anthrax may not be so against inhalational anthrax. The aim of this work was to optimize immunization with PA-FIS and to assess vaccine efficacy against inhalational anthrax. We assessed the immune response to recombinant anthrax PA from Bacillus anthracis (rPA)-FIS administered by various immunization protocols and the protection provided to mice and guinea pigs infected through the respiratory route with spores of a virulent strain of B. anthracis. Combined subcutaneous plus intranasal immunization of mice yielded a mucosal immunoglobulin G response to rPA that was more than 20 times higher than that in lung mucosal secretions after subcutaneous vaccination. The titers of toxin-neutralizing antibody and antispore antibody were also significantly higher: nine and eight times higher, respectively. The optimized immunization elicited total protection of mice intranasally infected with the virulent B. anthracis strain 17JB. Guinea pigs were fully protected, both against an intranasal challenge with 100 50% lethal doses (LD₅₀) and against an aerosol with 75 LD₅₀ of spores of the highly virulent strain 9602. Conversely, immunization with PA alone did not elicit protection. These results demonstrate that the association of PA and spores is very much more effective than PA alone against experimental inhalational anthrax.Bacillus anthracis is a gram-positive, aerobic, facultatively anaerobic, spore-forming, rod-shaped bacterium and is the etiologic agent of anthrax. B. anthracis resides in the soil as a dormant spore that is highly resistant to adverse conditions and can remain viable for years. The spore typically enters herbivores through ingestion; although anthrax is predominantly a disease of herbivores, humans can be infected through incidental exposure during handling of animals or animal products. In humans, the disease may take three forms—cutaneous, gastrointestinal, or pulmonary—depending on the site of entry. The most common human form is cutaneous anthrax, typically caused by spores infecting open wounds or skin abrasions. The mortality of cutaneous anthrax is near 20% if untreated (21). Gastrointestinal anthrax may in some cases extend to neuromeningitidis and generally leads to fatal systemic disease if untreated (5, 21). Naturally acquired pulmonary anthrax is very unusual. However, the mortality of pulmonary anthrax is almost 100% if not treated very early (80). Inhalational anthrax manifests as the rapid development of nonspecific, flulike symptoms that, if untreated, progress quickly to shock, respiratory distress, and death (21, 80).Inhaled spores are deposited in alveolar spaces where they are ingested by macrophages (39, 66) and by dendritic cells (DCs) (9, 15). Then, the intracellular spores germinate into nascent bacilli that escape from the macrophage, multiply extracellularly in the lymphatic system and spread into the bloodstream, where rapid multiplication continues (38, 39); alternatively, phagocytized spores are transported by migrating macrophages to the mediastinal and peribronchial lymph nodes, where they germinate into bacilli (66). DCs may be central to this step of the infection (15). Anthrax disease appears to result from a two-step process involving overwhelming bacterial replication and subsequent toxin production. Nevertheless, the fate of spores within macrophages, the resistance of macrophages to anthrax toxins and the role of macrophages in B. anthracis dissemination all remain controversial (19, 20, 38, 39, 83). An alternative mechanism has been recently described, suggesting that inhaled spores establish an initial infection in nasally associated lymphoid tissues where they germinate. The bacteria then disseminate first to the draining lymph nodes, then to the spleen and lungs, and finally to the blood (37).B. anthracis has two major virulence determinants. One is a tripartite protein complex toxin composed of lethal factor (LF), edema factor (EF), and protective antigen (PA) all encoded by plasmid pXO1. The other is antiphagocytic poly-γ-d-glutamic acid (γPDGA) capsule encoded by plasmid pXO2. EF and LF combine with PA to form the edema toxin (ET) and lethal toxin (LT), respectively, which both impair host immune defenses and probably act synergistically in vivo to cause edema formation and death (58, 75). The PA-LF/PA-EF complex is internalized by receptor-mediated endocytosis and, after acidification of the endosome, the toxin is translocated into the host cell cytosol, where it exerts cytotoxic effects (89). LT is a zinc metalloprotease that inactivates mitogen-activated protein kinase kinases, leading to toxic effects on susceptible macrophages (3, 18, 24, 54) and impairment of the bactericidal activity of alveolar macrophages, thus facilitating B. anthracis survival (35, 65). ET is a calmodulin-dependent adenylate cyclase that catalyzes the production of cyclic AMP from host ATP, perturbing water homeostasis, which in turn causes massive edema (55). ET is also cytotoxic in a cell-dependent manner and may contribute to the disease through directly killing cells, leading to tissue necrosis (79) and multiorgan failure, resulting in host death (28). LT and ET cooperatively inhibit activation of both DCs (14, 76) and T cells (57), thereby suppressing both the innate immune response and the priming of adaptive immune responses. Therefore, preventing either the entry of the toxin complex into the host cell or its translocation into the cytosol would make a major contribution to protection.The PDGA capsule is a poorly immunogenic polypeptide but seems to be vital for the dissemination of B. anthracis in the bodies of infected animals (12). The in vivo synthesis of capsule determines the outcome of infection (22, 49), and capsule degradation enhances both in vitro macrophage phagocytosis and neutrophil killing of encapsulated B. anthracis (68).The potential use of B. anthracis spores as a weapon of biological warfare or as inhaled weapons of bioterrorism has increased the need for a safe and effective vaccine to protect humans against inhalational anthrax (6, 31).The current United Kingdom licensed anthrax vaccine, anthrax vaccine precipitate, is an alum-precipitated filtrate of B. anthracis 34F2 Sterne strain culture consisting mainly of PA (77). The U.S. licensed anthrax vaccine absorbed (AVA/Biothrax) also consists mainly of PA, in this case extracted from cultures of the unencapsulated, toxin-producing strain of B. anthracis V770-NP1-R adsorbed onto aluminum hydroxide (33). Both vaccines contain small amounts of EF and LF and probably other components that presumably contribute to vaccine efficacy (33, 77, 88).These vaccines have the major disadvantage of inducing only a limited duration of protection and require frequent booster injections if sufficient immunity is to be maintained (32). Furthermore, such PA-based vaccines, acting on toxins, are less effective than live attenuated vaccines such as the Sterne strain, suggesting that additional antigens may contribute in a significant manner to protective immunity (4, 16, 42, 51, 59, 85).Various animal models have been used for testing the protective activities of vaccines against anthrax infection, including mice (10, 30, 86), rats (46), guinea pigs (10, 26, 46, 70), hamsters (27), rabbits (26, 50, 60, 61), and nonhuman primates (26, 40, 44, 60). These studies emphasize the large differences of protection between species. For instance, PA-based vaccines confer better protection to guinea pigs, rabbits, and nonhuman primates than to mice, probably because the γPDGA capsule is the primary virulence factor in mice (87). Indeed, many reports suggest that capsule antigen(s) (13, 47, 64, 67, 81) and spore antigen(s) (10, 16, 23) might confer additional protection. An immunodominant glycoprotein antigen of the spore surface (BclA) has been identified among the various surface proteins of the exosporium and may contribute to protective immunity (72, 74). Sera from animals immunized with living spores of the toxinogenic unencapsulated STI-1 strain of B. anthracis have been reported to express both antitoxin and antispore activities, the latter involving inhibition of spore germination, which was attributed by some authors to both anti-PA and anti-LF antibodies (73). Furthermore, PA-based vaccines induce antispore activity characterized by stimulation of phagocytosis of opsonized spores by murine macrophages in vitro and by inhibition of spore germination. As a consequence, anti-PA antibody-specific immunity may contribute to impeding the early stages of infection with B. anthracis spores (84).Brossier et al. demonstrated that the addition of formaldehyde-inactivated spores (FIS) of B. anthracis to PA antigen (PA-FIS) elicits total protection of mice and guinea pigs against subcutaneous (s.c.) challenge with a virulent B. anthracis strain (10). However, vaccines that are effective for the s.c. route of infection may not be so against the pulmonary route (30).Several studies have demonstrated that either live spore-based vaccines or PA-based vaccines may confer variable protection against different B. anthracis strains and isolates in both mice and guinea pigs (26, 43, 51, 82, 85). Therefore, we used two different B. anthracis challenge strains in our study, namely, strains 9602 and 17JB from the Institut Pasteur collection. Although both strains are encapsulated and toxinogenic (cap⁺ tox⁺), harboring both pXO1 and pXO2 plasmids, they differ in virulence, as shown by the 50% lethal doses (LD₅₀) (s.c. route), estimated to be about 50 and 500 spores per mouse, respectively (10). Strain 9602 is as virulent as the Ames strain (10, 43); strain 17JB (the atypical Pasteur vaccine strain 2-17JB (78), harboring both pXO1 and pXO2 (cap+ tox+), is very similar to the so-called “Carbosap” strain used in Italy for immunization against ovine and bovine anthrax (25). It has residual pathogenicity characteristics that cause death in mice and guinea pigs but expresses no virulence in rabbits (25). Adone et al. demonstrated that the attenuation of the Carbosap vaccine strain is not due to the lack of virulence genes (cya, lef, and pagA), of regulatory genes (atxA and pagR), or of the gerX operon involved in germination within macrophages, or to divergence of the sequences of these genes from those of a wild-type virulent B. anthracis strain (1). Indeed, sophisticated advanced molecular analysis has been unable to identify the genetic differences accounting for differences in virulence between Carbosap and virulent strains (48).There are various possible causes of these differences in virulence and pathogenesis, including (i) involvement of unknown virulence factors and/or mechanisms involved in attenuation, (ii) differences in expression and activity of the known virulence factors and their regulators (48), and (iii) differences in pXO2 plasmid copy number (17). Nevertheless, like the Vollum strain, 17JB remains a relevant model for the study of vaccine efficacy: it is less pathogenic than wild-type strains such as 9602 or Ames but is nevertheless cap⁺ tox⁺.In summary, despite obvious efficacy in nonhuman primates, the currently licensed anthrax vaccines have shortcomings, such as a limited duration of protection and the need for frequent booster injections. Moreover, trace amounts of LF, EF, and probably other components are likely to have contributed to the efficacy of the vaccine in the reported studies. For instance, AVA provides partial protection in a guinea pig model of inhalational anthrax, whereas a recombinant anthrax PA from B. anthracis (rPA)-based vaccine elicits no protection (53). Furthermore, PA-based vaccines may confer variable protection against different B. anthracis strains and isolates, and large differences in the level of protection afforded are observed between animal species. These limitations have stimulated interest in the development of improved anthrax vaccines. The data discussed above suggest that other antigens in addition to PA are required for full protection.The aim of the present study was to optimize the PA-FIS vaccine immunization protocol so as to elicit protection against inhalational anthrax in an experimental model of lung infection. We assessed the systemic and mucosal immune response to PA-FIS in mice and guinea pigs, immunized either through the s.c. or the intranasal (i.n.) route or both. Second, we assessed the protection afforded in an experimental model of inhalational anthrax of mice and guinea pigs infected by nasal instillation or an aerosol.     

Risk factors for pancreatic cancer: case-control study

OBJECTIVES: Although cigarette smoking is the most well-established environmental risk factor for pancreatic cancer, the interaction between smoking and other risk factors has not been assessed. We evaluated the independent effects of multiple risk factors for pancreatic cancer and determined whether the magnitude of cigarette smoking was modified by other risk factors in men and women. METHODS: We conducted a hospital-based case-control study involving 808 patients with pathologically diagnosed pancreatic cancer and 808 healthy frequency-matched controls. Information on risk factors was collected by personal interview, and unconditional logistic regression was used to determine adjusted odds ratios (AORs) by the maximum-likelihood method. RESULTS: Cigarette smoking, family history of pancreatic cancer, heavy alcohol consumption (>60 mL ethanol/day), diabetes mellitus, and history of pancreatitis were significant risk factors for pancreatic cancer. We found synergistic interactions between cigarette smoking and family history of pancreatic cancer (AOR 12.8, 95% confidence interval [CI] 1.6-108.9) and diabetes mellitus (AOR 9.3, 95% CI 2.0-44.1) in women, according to an additive model. Approximately 23%, 9%, 3%, and 5% of pancreatic cancer cases in this study were related to cigarette smoking, diabetes mellitus, heavy alcohol consumption, and family history of pancreatic cancer, respectively. CONCLUSIONS: The significant synergy between these risk factors suggests a common pathway for carcinogenesis of the pancreas. Determining the underlying mechanisms for such synergies may lead to the development of pancreatic cancer prevention strategies for high-risk individuals.     

Extrahepatic Bile Duct Adenocarcinoma: Patients at High-Risk for Local Recurrence Treated with Surgery and Adjuvant Chemoradiation Have an Equivalent Overall Survival to Patients with Standard-Risk Treated with Surgery Alone

Background  Patients with resected extrahepatic bile duct adenocarcinoma who have microscopically positive resection margins and/or pathologic locoregional nodal involvement (R1pN1) have a high-risk of locoregional recurrence, and therefore, we advocate the use of adjuvant chemoradiation. To evaluate the safety and effectiveness of this treatment, we compared survival and side effects outcomes between such patients and patients with negative resection margins and pathologically negative nodes (R0pN0) who did not receive adjuvant treatment. Methods  Between 1984 and 2005, 65 patients were treated with curative-intended resection for extrahepatic bile duct adenocarcinoma. Patients with tumors arising in the gallbladder and periampullary region were excluded. Pathology and diagnostic images were centrally reviewed. Overall survival and locoregional recurrence outcomes for patients with standard-risk R0pN0 (surgery alone, or S group, n = 23) were compared with those of patients with high locoregional recurrence risk, R1 and/or pN1 (R1pN1) status who received adjuvant chemoradiation (S-CRT group, n = 42). Results  The median follow-up for the entire group was 31 months. Patients in the S-CRT and S groups had a similar 5-year overall survival (36% vs. 42%, P = .6) and locoregional recurrence (5-year rate: 38% vs. 37%, P = .13). In the S-CRT group, three patients (7%) experienced an acute (grade 3 or more) side effect. Conclusions  Our finding of a lack of a survival difference between the S and S-CRT groups suggests that for patients with extrahepatic bile duct adenocarcinoma at high risk for locoregional recurrence (i.e., R1 resection or pN1 disease), adjuvant chemoradiation provides an equivalent overall survival despite of these worse prognostic features.     

Twenty seven years of experience in pediatric liver transplantation in Strasbourg: focus on the ex situ split techniques

INTRODUCTION: Despite the well-known controversies about split-liver procedures, since 1979 we have utilized an ex situ instead of an in situ technique because of its feasibility. However, we sought to prove the equality of the results of these two procedures. Herein, we have presented our experience after 27 years' follow-up. MATERIALS AND METHODS: Between March 1979 and June 2006, we transplanted 84 livers in 67 pediatric recipients including 37 ex situ split livers implanted into 28 patients. RESULTS: We recorded demographic characteristics, transplantation, and retransplantation indications, age difference between donors and recipients, comorbidities, cold ischemia times, surgical times and complications, graft/recipient body weight ratios, organ recovery times, and overall survivals after 1, 5, and 15 years follow-up. We have herein reported 1, 5, and 15 years of patient versus organ survivals of 88.9.1%, 84.5%, 62.1% versus 78.6%, 74.2%, 57.4%, respectively. CONCLUSION: We have concluded that an ex situ split liver may be a valid alternative to in situ techniques to achieve good grafts for pediatric transplantation.     

Solitary Metastasis From Cutaneous Melanoma to the Liver: Resection by Extended Left Hepatectomy (Trisegmentectomy) With Clearance of Tumor From the Portal Vein

A 61-year-old woman presented with low grade fever and an epigastric mass eight years following resection of a stage Clark IV infraclavicular cutaneous melanoma followed by axillary node dissection. Investigations revealed a tumor in segment II, III, IV and V of the liver and a thrombus involving the main portal vein. Liver resection with extended left hepatectomy (left trisegmentectomy) and portal vein thrombectomy is reported.     

First-in-Human Phase 1b Trial of Quinacrine Plus Capecitabine in Patients With Refractory Metastatic Colorectal Cancer

BackgroundQuinacrine plus a fluoropyrimidine has in vivo efficacy against metastatic colorectal cancer (mCRC). This phase 1b trial evaluated the combination of quinacrine plus capecitabine in patients with treatment-refractory mCRC.Patients and MethodsUsing a modified Simon accelerated titration design, adults with treatment-refractory mCRC were treated with capecitabine 1000 mg/m² twice daily for 14/21-day cycle, and escalating doses of quinacrine 100 mg daily, 100 mg twice daily, and 200 mg twice daily for 21 days. The primary endpoint was identifying the maximum tolerated dose, determining tolerability and safety. In an expansion cohort, it was overall response rate and time to tumor progression (TTP).ResultsTen patients (median age of 60 years) were treated in phase 1b. The first 2 quinacrine dosing levels were well tolerated. Dose-limiting toxicities were seen in 3 patients treated with quinacrine 200 mg twice daily. Five additional patients tolerated quinacrine 100 mg twice daily without further dose-limiting toxicities, thus establishing the maximum tolerated dose. Seven additional expansion-cohort patients enrolled onto the study before quinacrine manufacturing ceased within the United States. Five patients experienced stable disease, 1 partial response, and 10 disease progression. Median TTP overall was 2.12 months and median overall survival 5.22 months for the 17 patients.ConclusionCapecitabine and quinacrine can be safely administered at the maximum tolerated dose of capecitabine 1000 mg/m² by mouth twice daily on days 1-14 and quinacrine 100 mg by mouth twice daily on days 1-21 of a 21-day cycle in mCRC patients. Although the expansion study was halted early, TTP was in line with other studies of refractory mCRC, suggesting activity of this regimen in heavily pretreated patients.