Lipopolysaccharide as an Antigen Target for the Formulation of a Universal Vaccine against Escherichia coli O111 Strains

A promising approach to developing a vaccine against O111 strains of diarrheagenic Escherichia coli that exhibit different mechanisms of virulence is to target either the core or the polysaccharide chain (O antigen) of their lipopolysaccharide (LPS). However, due to structural variations found in both these LPS components, to use them as antigen targets for vaccination, it is necessary to formulate a vaccine able to induce a humoral immune response that can recognize all different variants found in E. coli O111 strains. In this study, it was demonstrated that, despite differences in composition of oligosaccharide repeat units between O111ab and O111ac LPS subtypes, antibodies against one O111 subtype can recognize and inhibit the adhesion to human epithelial cells of all categories of O111 E. coli (enteropathogenic E. coli [EPEC], enterohemorrhagic E. coli [EHEC], and enteroaggregative E. coli [EAEC]) strains regardless of the nature of their flagellar antigens, mechanisms of virulence, or O111 polysaccharide subtypes. These antibodies were also able to increase the clearance of different strains of O111 E. coli by macrophages. PCR analyses of the pathways involved in O111 LPS core biosynthesis showed that all EAEC strains have core type R2, whereas typical EPEC and EHEC have core type R3. In contrast, atypical EPEC strains have core types R2 and R3. In summary, the results presented herein indicate that the O111 polysaccharide and LPS core types R2 and R3 are antigen targets for panspecific immunotherapy against all categories of O111 E. coli.Pathogenic strains of O111 Escherichia coli exist as three distinct categories of diarrheagenic organisms, namely, enteropathogenic E. coli (EPEC; typical and atypical), enterohemorrhagic E. coli (EHEC), and enteroaggregative E. coli (EAEC) (7). In developing countries, diarrhea induced by these pathogens is a serious illness that inflicts a huge health and economic burden on the population (46, 48). Despite the fact that sanitation and clean water can markedly reduce the cases of diarrhea in areas of endemicity, surveillance studies have demonstrated that in Latin America alone more than 80% of the population has no access to sewage systems or treated water (44). Different serotypes of Shiga toxin-producing E. coli pathogens (O111:H⁻, O111:H8, and O111:H2) are also a public health problem in developed countries worldwide, where they have been responsible for outbreaks of bloody diarrhea and cases of hemolytic-uremic syndrome (HUS) (4, 12, 14, 21, 28, 32, 35, 55). One of the worst outbreaks of O111 E. coli happened in August 2008 in Oklahoma, where 341 people become ill, 70 people were hospitalized, 17 people developed HUS, and 1 person died (5, 8). In addition, other pathogens such as Salmonella enterica subsp. enterica serovar Adelaide and Salmonella enterica subsp. enterica serovar 50:z:e,n,x also have the same lipopolysaccharide (LPS) polysaccharide structure as that found in O111 E. coli (29).Because of the impact that O111 E. coli strains have on public health, a lot of effort has been devoted to developing a safe, cheap, and effective vaccine to prevent diarrheagenic diseases caused by these pathogens.The best approach to constructing a vaccine capable of protecting against a wide range of different strains of O111 E. coli is to target the LPS polysaccharide chain (O antigen), since 75% of the outer membrane of all Gram-negative bacteria is covered by LPS (38, 50). This approach is supported by the fact that conjugated vaccines against polysaccharides have been used successfully against polysaccharide-encapsulated organisms such as Streptococcus pneumoniae and Haemophilus influenzae type b in clinical practice (42). However, to use the O111 polysaccharide chain as an antigen target for the construction of a universal vaccine against enteric O111 E. coli pathogens, the antigenic variation of O111 subtypes between different E. coli strains has to be taken into account (7, 33, 59). In addition, although the O111 polysaccharides that compose their capsules are identical to the ones present on their external membranes (17, 53, 54), it has been demonstrated by Goldman and coworkers that the capsules of O111 bacteria are poorly recognized by antibodies raised against O111 LPS derived from the bacterial membrane (17), indicating that immunization with capsulated bacteria induces antibody responses different from those induced by immunization with noncapsulated bacteria.In addition, the O111 E. coli strains can be either naked or capsulated, although the O111 polysaccharides that compose their capsules are identical to the ones present on their external membranes, except for the absence of a lipid A core (17, 53, 54).The LPS core can also be targeted for vaccination or immunotherapy (11, 19, 39). It is not considered a virulence factor, although its involvement in bacterial adhesion has been reported (24). Structural variations are also found in the external part of the LPS core (37), and they have to be considered in order to generate antibodies capable of identifying all antigenic variants encountered within O111 bacteria.Another element of the humoral immune response involved in clearance of pathogens is the complement system, which, independently of antibody, can be activated by pathogens in the initial stages of infection and, by itself, can kill pathogens directly. However, it is not effective in recognizing or eliminating all bacteria in samples (3, 30, 43, 45). The complement system can also promote bacterial uptake and destruction by macrophages by interacting with both the pathogen and the complement receptors present on the macrophage membrane. However, when complement activation is not enough to promote bacterial killing by macrophages, antibodies are required (25, 26, 34).To investigate whether the O111 LPS polysaccharide of E. coli is a good antigen candidate for the formulation of a universal vaccine capable of preventing infection by O111 pathogens, electrophoretic, molecular, serological, and immunological analyses were conducted in order to determine whether antibodies against O111 polysaccharides can recognize O111 EHEC, EPEC, and EAEC, can inhibit their adhesion to human epithelial cells, and can stimulate their clearance by macrophages.In addition, the compositions of the cores of 73 samples of all categories of O111 bacteria were characterized by PCR analysis of the enzymes responsible for the biosynthesis of all five types of LPS core: R1, R2, R3, R4, and K12.     

A tolC Mutant of Francisella tularensis Is Hypercytotoxic Compared to the Wild Type and Elicits Increased Proinflammatory Responses from Host Cells

The highly infectious bacterium Francisella tularensis is a facultative intracellular pathogen and the causative agent of tularemia. TolC, which is an outer membrane protein involved in drug efflux and type I protein secretion, is required for the virulence of the F. tularensis live vaccine strain (LVS) in mice. Here, we show that an LVS ΔtolC mutant colonizes livers, spleens, and lungs of mice infected intradermally or intranasally, but it is present at lower numbers in these organs than in those infected with the parental LVS. For both routes of infection, colonization by the ΔtolC mutant is most severely affected in the lungs, suggesting that TolC function is particularly important in this organ. The ΔtolC mutant is hypercytotoxic to murine and human macrophages compared to the wild-type LVS, and it elicits the increased secretion of proinflammatory chemokines from human macrophages and endothelial cells. Taken together, these data suggest that TolC function is required for F. tularensis to inhibit host cell death and dampen host immune responses. We propose that, in the absence of TolC, F. tularensis induces excessive host cell death, causing the bacterium to lose its intracellular replicative niche. This results in lower bacterial numbers, which then are cleared by the increased innate immune response of the host.Francisella tularensis is the etiological agent of tularemia. F. tularensis is classified as a category A agent of bioterrorism by the U.S. Centers for Disease Control and Prevention (http://emergency.cdc.gov/agent/agentlist-category.asp) due to its low infectious dose, ease of aerosol dissemination, and capacity to cause high morbidity and mortality (19). There are two clinically relevant subspecies of F. tularensis: subsp. tularensis, which is extremely pathogenic in humans, and subsp. holarctica, which causes a less severe clinical presentation (48). The most severe form of the disease is pneumonic tularemia caused by the inhalation of aerosolized F. tularensis subsp. tularensis (19). The F. tularensis subsp. holarctica-derived live vaccine strain (LVS) was used for many years as the vaccination against tularemia. However, the basis for its attenuation is unknown, and it is no longer in use as a vaccine (46). The LVS is highly virulent in mice, where it causes a disease closely resembling human tularemia (30). These features make the LVS an important model for the study of tularemia. An additional Francisella species, F. novicida, causes disease only in immunocompromised individuals. F. novicida, like the LVS, is highly virulent in mice and widely used as a model of tularemia (20).F. tularensis is a Gram-negative, facultative intracellular pathogen (50). Although factors important for the virulence of F. tularensis are beginning to be identified, the molecular mechanisms behind the extreme pathogenicity of this organism still are largely unknown. In vivo, F. tularensis is a stealth pathogen, evading host cell defenses and dampening host proinflammatory responses. F. tularensis produces an unusual lipopolysaccharide that has low toxicity and does not activate host cells in a TLR4-dependent manner (4, 22). A critical aspect of the pathogenesis of F. tularensis is its ability to escape the phagosome and replicate within the cytosol of a variety of host cells, including both murine and human macrophages and dendritic cells (2, 3, 16, 25, 49). Although F. tularensis does have an extracellular phase (24), it is thought that cytosolic replication allows the bacteria to grow to large numbers while avoiding detection by the host immune system.Host cells respond to F. tularensis invasion by inducing cell death pathways, including apoptosis and pyroptosis (32, 38). In the intrinsic apoptotic pathway, cytochrome c is released from mitochondria into the cytosol, leading to caspase-9 activation and ultimately to the activation of effector caspases such as caspase-3 and -7 (10). In pyroptosis, caspase-1 is activated through the inflammasome complex, resulting in the release of proinflammatory cytokines such as interleukin-1ß (IL-1ß) (6, 32). Lai and coworkers demonstrated that the infection of murine J774 macrophage-like cells with the LVS activated the intrinsic apoptotic pathway as early as 12 h postinfection. Activated caspase-3, but not caspase-1, was detected in the infected cells (38). In contrast, Mariathasan et al. found that the infection of preactivated murine peritoneal macrophages by either the LVS or strain U112 (F. novicida) triggered pyroptosis and the release of IL-1ß (42). In both studies, the induction of cell death was dependent upon the bacteria escaping the phagosome and initiating cytosolic replication. Weiss and colleagues isolated mutants of strain U112 that were attenuated in vivo and caused increased cell death in tissue culture compared to that caused by wild-type U112 (53). This suggests that although host cells initiate death pathways in response to F. tularensis infection, the bacteria has the ability to actively reduce cell death, and this is important for virulence.In addition to triggering death pathways, host cells respond to invading bacteria by mounting a proinflammatory response to alert neighboring cells of the impending bacterial threat (17). However, F. tularensis has been shown to actively suppress these innate host responses. Telepnev and coworkers showed that the LVS disrupted toll-like receptor signaling and blocked the secretion of the proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1ß by murine and human macrophages (51, 52). Similarly, Bosio and colleagues showed that the LVS inhibited the innate immune response of murine pulmonary dendritic cells to bacterial ligands, and the infection of mice with the fully virulent Schu4 strain (F. tularensis subsp. tularensis) caused an overall state of immunosuppression in the lungs (8, 9).The genome analysis of F. tularensis identified only a few potential virulence factors, suggesting that the bacterium uses novel factors to achieve its high level of pathogenicity (40). Unique to F. tularensis is a 33.9-kb region of DNA termed the Francisella pathogenicity island (FPI) (29, 40, 45). The FPI encodes genes that are essential for intracellular survival and virulence, including iglABCD and pdpABCD (45). F. tularensis lacks type III and IV secretion systems, which is surprising considering its intracellular nature. These secretion systems commonly are used by intracellular pathogens to deliver effector proteins inside host cells to manipulate host cell responses (14, 26). F. tularensis does contain genes encoding a type IV pilus biogenesis system that also functions in the secretion of soluble proteins by a type II-like mechanism and that are important for virulence (12, 31, 54). Finally, F. tularensis appears to contain a functioning type I secretion system that is critical for pathogenesis (28).Type I secretion systems function in the secretion of a variety of toxins and other virulence factors directly from the cytoplasm to the extracellular milieu in a single energized step (33, 37). The type I system consists of three separate components: an outer membrane channel-forming protein, a periplasmic adaptor or membrane fusion protein, and an inner membrane pump that typically belongs to the ATP-binding cassette family. The TolC protein of Escherichia coli, which functions in hemolysin secretion, is the prototypical outer membrane channel component (37). In addition to protein secretion, TolC functions in the efflux of small noxious molecules, conferring multidrug resistance (37). F. tularensis contains three TolC paralogs, TolC, FtlC, and SilC, with TolC and FtlC exhibiting significant homology to the E. coli TolC protein (28, 35). In a previous study we created tolC and ftlC deletion mutants in the F. tularensis LVS (28). We found that both TolC and FtlC participate in multidrug resistance in F. tularensis, but only the ΔtolC mutant was attenuated for virulence in mice by the intradermal route. Thus, tolC is a critical virulence factor of F. tularensis and likely functions in type I secretion in addition to multidrug efflux.Here, we delineate the molecular mechanisms behind the attenuation of the LVS ΔtolC mutant in mice infected by both the intradermal and intranasal routes. In vivo organ burden assays revealed that the ΔtolC strain is decreased for the bacterial colonization of liver, spleen, and most prominently, lungs. In vitro experiments revealed that the ΔtolC mutant is hypercytotoxic to murine macrophages, causing increased apoptosis via a mechanism involving caspase-3 but not caspase-1. In addition, the LVS ΔtolC mutant was hypercytotoxic toward human macrophages and elicited the significantly increased secretion of the proinflammatory chemokines CXCL8 (also known as IL-8) and CCL2 (also known as monocyte chemoattractant protein [MCP-1]). Taken together, these data demonstrate a critical role for TolC, likely via a TolC-secreted toxin(s), in the successful intracellular lifestyle of F. tularensis, its ability to evade host innate immune responses, and its overall virulence.     

Age-related changes in cardiac autonomic control during sleep

Aging is commonly associated with decreased sleep quality and increased periodic breathing (PB) that can influence heart rate variability (HRV). Cardiac autonomic control, as inferred from HRV analysis, was determined, taking into account the sleep quality and breathing patterns. Two groups of 12 young (21.1 +/- 0.8 years) and 12 older (64.9 +/- 1.9 years) volunteers underwent electroencephalographic, cardiac, and respiratory recordings during one experimental night. Time and frequency domain indices of HRV were calculated in 5-min segments, together with electroencephalographic and respiratory power spectra. In the elderly, large R-R oscillations in the very-low frequency (VLF) range emerged, that reflected the frequency of PB observed in 18% of the sleep time. PB occurred more frequently during rapid eye movement sleep (REM) sleep and caused a significant (P < 0.02) increase in the standard deviation of normal R-R intervals (SDNN) and absolute low-frequency (LF) power. With normal respiratory patterns, SDNN, absolute VLF, LF, and high frequency (HF) power fell during each sleep stage (P < 0.01) compared with young subjects, with no significant sleep-stage dependent variations. An overall decrease (P < 0.01) in normalized HF/(LF + HF) was observed in the elderly, suggesting a predominant loss of parasympathetic activity which may be related to decreased slow-wave sleep duration. These results indicate that two distinct breathing features, implying different levels of autonomic drive to the heart, influence HRV in the elderly during sleep. The breathing pattern must be considered to correctly interpret HRV in the elderly.     

A LysR-type transcriptional regulator in Burkholderia cenocepacia influences colony morphology and virulence

Burkholderia cenocepacia strain K56-2 typically has rough colony morphology on agar medium; however, shiny colony variants (shv) can appear spontaneously. These shv all had a minimum of 50% reduction in biomass formation and were generally avirulent in an alfalfa seedling infection model. Three shv-K56-2 S15, K56-2 S76, and K56-2 S86-were analyzed for virulence in a chronic agar bead model of respiratory infection and, although all shv were able to establish chronic infection, they produced significantly less lung histopathology than the rough K56-2. Transmission electron microscopy revealed that an extracellular matrix surrounding bacterial cells was absent or reduced in the shv compared to the rough wild type. Transposon mutagenesis was performed on the rough wild-type strain and a mutant with an insertion upstream of ORF BCAS0225, coding for a putative LysR-type regulator, exhibited shiny colony morphology, reduced biofilm production, increased N-acyl homoserine lactone production, and avirulence in alfalfa. The rough parental colony morphotype, biofilm formation, and virulence in alfalfa were restored by providing BCAS0225 in trans in the BCAS0225::pGSVTp-luxCDABF mutant. Introduction of BCAS0225 restored the rough morphotype in several shv which were determined to have spontaneous mutations in this gene. In the present study, we show that the conversion from rough wild type to shv in B. cenocepacia correlates with reduced biofilm formation and virulence, and we determined that BCAS0225 is one gene involved in the regulation of these phenotypes.     

Training medical students in community health: a novel required fourth-year clerkship at the University of Rochester.

In 2004, community health became the fourth mission of the University of Rochester Medical Center, along with education, clinical care, and research. In that same year, a novel clerkship was added to the fourth-year curriculum that focuses on the "practice" of community health and preventive medicine. The goal is to offer intensive experiential training to develop skills in community health improvement by partnering with community agencies involved in health promotion and disease prevention. The learning objectives addressed include community health assessment, risk behavior change, assurance of personal health services, advocacy and policy change, environmental interventions, community organization and partnership building, and program evaluation. The clerkship involves three full days of didactic instruction at the beginning of a four-week period of program development and implementation. Each student chooses a project that focuses on a specific target population, then designs it and incorporates public health knowledge, skills, and attitudes learned during the didactic component. Course directors then mentor students during project implementation. Students can begin "longitudinal" experiences in their first or second years to fold into the required clerkship. Innovations include a novel Advocacy and Policy Change module, a highly rated Cultural Determinants of Health lecture, and a resource-based course Web site. The clerkship was initially offered as an elective, and it has since become a required course. In the clerkship to date, 340 students have launched hundreds of community-level interventions within various settings locally, nationally, and internationally. Evaluation efforts to date indicate that the clerkship has been received favorably by both faculty and students.     

Evidence for a colorectal cancer susceptibility locus on chromosome 3q21-q24 from a high-density SNP genome-wide linkage scan

To identify a novel susceptibility gene for colorectal cancer (CRC), we conducted a genome-wide linkage analysis of 69 pedigrees segregating colorectal neoplasia in which involvement of known loci had been excluded, using a high-density single nucleotide polymorphism (SNP) array containing 10,204 markers. Multipoint linkage analyses were undertaken using both non-parametric (model-free) and parametric (model-based) methods. After the removal of SNPs in strong linkage disequilibrium, we obtained a maximum non-parametric linkage statistic of 3.40 (P=0.0003) at chromosomal region 3q21-q24. The same genomic position also yielded the highest multipoint heterogeneity LOD (HLOD) score under a dominant model (HLOD=3.10, genome-wide P=0.038) with 62% of families linked to the locus. We provide evidence for a novel CRC susceptibility gene. Further studies are needed to confirm this localization and to evaluate the contribution of this locus to disease incidence.     

Germline mutations in RAD51, RAD51AP1, RAD51B, RAD51C,RAD51D, RAD52 and RAD54L do not contribute to familial chronic lymphocytic leukemia.

While familial predisposition to B-cell chronic lymphocytic leukemia (CLL) is well recognized no gene which when mutated in the germline has been unambiguously shown to confer susceptibility to the disease. An approach based on mutation screening methods targeted to coding regions of candidate genes offers an attractive strategy for the identification of rare disease-causing alleles. The RAD genes participate in the cellular response to DNA double strand breaks, detecting DNA damage, activating cell cycle checkpoints and apoptosis. Defects in members of these genes are linked to increased chromosomal instability and in lymphoma predisposition, thereby representing strong candidate susceptibility genes a priori. To examine this proposition we screened 75 familial CLL probands for germline mutations in this set of genes. No overt pathogenic mutations were identified. These findings indicate that germline mutations in RAD51, RAD51AP1, RAD51L1, RAD51L3, RAD52 and RAD54L are unlikely to be causal of an inherited predisposition to CLL.     

Consensus guidelines for the management of radiation dermatitis and coexisting acne-like rash in patients receiving radiotherapy plus EGFR inhibitors for the treatment of squamous cell carcinoma of the head and neck

Background: Radiation dermatitis occurs to some degree in mostpatients receiving radiotherapy, with or without chemotherapy.Patients with squamous cell carcinoma of the head and neck (SCCHN)who receive radiotherapy in combination with epidermal growthfactor receptor (EGFR) inhibitors, such as cetuximab, may developa characteristic acne-like rash in addition to dermatitis. Design: An advisory board of 11 experienced radiation oncologists,medical oncologists and dermatologists discussed the managementoptions for skin reactions in patients receiving EGFR inhibitorsand radiotherapy for SCCHN. Skin toxicity was categorised accordingto the National Cancer Institute—Common Terminology Criteriafor Adverse Events (version 3) grading. Results: Both general and grade-specific approaches for themanagement of dermatitis in this patient group are presented.It was concluded that where EGFR inhibitor-related acne-likerash and dermatitis coexist within irradiated fields, managementshould be based on the grade of dermatitis: for grade 1 (orno dermatitis), treatment recommendations for EGFR-related acne-likerash outside irradiated fields should be followed; for grades2 and above, treatment recommendations for dermatitis were proposed. Conclusions: This paper presents comprehensive consensus guidelinesfor the treatment of dermatitis in patients with SCCHN receivingEGFR inhibitors in combination with radiotherapy. Key words: cetuximab, EGFR inhibitors, radiation dermatitis, radiotherapy, skin reactions, squamous cell carcinoma of the head and neck Received for publication June 26, 2007. Revision received July 12, 2007. Accepted for publication July 12, 2007.