Project ID: FORGEN II IS3
Abstract: Aim of the Project:
Development of new live vaccines consisting of the benevolent Escherichia coli strain DSM6601 and inactivated virulence genes of pathogenic bacteria. The resulting vaccines will be produced by our cooperation partner "Ardeypharm" GmbH.
The Project: Certain E. coli strains, pathogenic for humans, colonize farm animals without causing symptoms. These include enterohemorrhagic E. coli (EHEC) causing sometimes fatal disease also in Germany and enterotoxigenic E. coli responsible for diarrhea in travellers and children in third world countries. Both E. coli groups express adhesins and toxins, which constitute main virulence factors. The responsible genes will be molecularly cloned and mutagenized in order to loose virulence capacitiy but retain immunogenicity. The mutated genes will be expressed in E. coli strain DSM6601. The introduction of genes encoding an invasion system and/or the Listeria monocytogenes hemolysin will allow the vaccine strains to deliver the antigens into endosomes and/or the cytoplasm of host cells. This targeted delivery allows the induction of defined immune responses. Vaccination with the recombinant DSM6601 strains of e.g. cattle will block colonization of these animals by the corresponding bacterial pathogens. Additionally, the Mip antigen from Legionella pneumophila and Chlamydia will also be introduced into DSM6601 to generate vaccines against these intracellular pathogens. This project will not only produce new vaccines but will help to develop new strategies for vaccine design.
Fibromyalgia and Intestinal Infection: The Bacteriophages Connection ImmuneSupport.com
By Theophil Hey, M.D.
Based on the scientific results of twelve years of continuous investigation we now regard as proven that a connection exists between Primary Fibromyalgia Syndrome (PFS) and evidence of bacteriophages (specific E. coli phages) found in the stool of patients.
The virology department of the Medical College in Hanover (MHH) used electron- microscopes to detect bacteriophages (viruses) in the stool of more than 80% of patients examined. By comparison, they were detected in only 12 - 13% of the control group. Scientifically speaking, this result is deemed to be of great scientific significance.
Bacteriophages (viruses) infect their specific host cell (in this case the E. coli bacteria) and cause the E. coli bacteria to produce new viruses via the viral genes.
Up to 400 new viruses develop in this way inside the E. coli bacteria. After the E. coli bacteria has practically surrendered its cell contents for the production of the virus it then dies (lytic virus reproduction) and bursts. A virus “brood” results. A number of the released viruses find new, not yet infected E. coli bacteria, infects them and the whole process begins anew. Medically significant and the cause of the patients symptoms are the particles of the sheaths released during the process of E-coli lysis (the death of the bacteria) - the lipopolysaccharides with their active component Lipid A - which represent highly potent endotoxins.
This means that primary fibromyalgic syndrome (often found in combination with chronic fatigue syndrome and irritable colon) is to be assessed primarily as an infection of the intestine - specific bateriophages infect their specific host cells (in this case E coli) - secondly, through the release of endotoxins, as intoxication locally (reaction of the bowels) and in the blood circulation after absorption through the intestinal walls.
If no significant change in the bacterial colonisation of the bowels comes about, this process can result in the patient experiencing an alternating course of the disease lasting months and years. To minimise or even eliminate the symptoms we must attempt to keep the virus away from its host cell (E. coli), or, at least, to minimise it permanently.
1. Cleansing of the bowels:
Day 1: e.g., salinic purgatives
Day 2 - 6: colistin sulphate ( - Diarönt mono 4x2.000.000 I.U.) together with Nystatin (4x1.000.000 I.U.)
2. Symbiosis management: -Saccharomyces boulardii (possibly permanently), 250mg 2x1 before meals, e.g., Perocur forte -Lactobacillus acid. (e.g. Paidoflor 2x1 after meals) -Lactulose ( e.g. Bifiteral 1 measure mornings)
3. Prevention of new infection: no raw meat (everything boiled or well-done; boiled salamis, boiled ham; no fast-food) no fresh milk (not been pasteurized), no cheese made from fresh milk, no raw vegetables or salad which have been grown in natural or liquid manure.
A low E-coli count means: the viruses can only infect a small number of E. coli and so cause very few to die. Consequently low amounts of endotoxins (poisons) are released. On the other hand: mass colonisation of the bowels by E-coli bacteria at the same time as their specific bateriophages (viruses) are present can result in a massive release of poisons and exceedingly unpleasant symptoms.
We are ready at all times to help if you have further questions, but must point out that an immediate reply is not always possible.
The pathogenesis of the primary fibromyalgic syndrome
Association of primary fibromyalgic syndrome (PFS) with evidence of bacteriophages in the stool. T. Hey, General Practitioner; A. Breull; G.C.Fischer, Dept. of General Medicine; W. Verhagen, Dept. of Virology; Med. Hochschule Hannover
The symptoms of PFS have been the subject of studies by numerous authors in the past ten years; they have repeatedly been described in detail, questions have been asked as to possible causes, many possible associations discussed and more or less effective therapy methods recommended. PFS represents a daily new challenge for us doctors - everyday we are faced with patients suffering from the unpleasant symptoms. They come hoping for an explanation - and for relief from their complaint.
This was the reason for me to make use of all possibilities available to me in an investigation into the pathogenesis of the symptoms. It is well known that PFS is a follow-up diagnosis. BSG, blood count, RF, CRP, CK as well as internal, orthopaedic and x-ray diagnosis show no evidence of a pathological substrate. It is merely the tender-points (ACR model) which can be clinically confirmed by the examiner as being typically symptomatic.
No more was known about the complaint up to this time. There was no known cause for this illness. All attempted treatments were treatment of the symptoms using analgesics, anti-rheumatic agents, tri-cyclic anti-depressives and exhaustive clarification of the course of the illness involving a great amount of patient-time.
Patients in my survey were first subjected to the same diagnostic steps before the question of a virus genesis was broached. Antibody tests were carried out to search for Coxackie B viruses, Herpes simplex viruses, Varicella zoster viruses, Entero viruses, Adeno viruses as well as Epstein-Barr viruses but no evidence of an association with the complaint was to be found.
Attempts to breed the virus taken from the blood, urine, stool and pharyngeal wash on egg albumin also failed to produce any positive results. I consulted with the department for virology in the MHH, Hanover and they recommended as a last diagnostic possibility examination of the stool under electron microscopes. This diagnostic step brought evidence of vast numbers of bacteriophages in the stool of the first 5 patients!
This represented the first objectively demonstrable substrate above and beyond the clinical symptoms which was common to all patients. The question now was: Is it at all theoretically possible for bacteriophages to harm human body cells? Can other mechanisms - triggered by the presence of bacteriophages - strain the human organism? The bacteriophages detected were quite definitely T-phages - i.e., specific E-coli phages. Which means that they cannot harm the human cell.
When this virus has reached its specific host cell and infected it, there follows a multiplication of the virus - (lytic phase) or lysogenesis occurs, i.e., the phage genom is attached to the E-coli DNA and passed on with every splitting of the E-coli. Through factors as yet unknown to us this passive virus phase can change back into the vegetative - i.e., lytic - form and so pass into the afore-mentioned multiplication process again. The E-coli disintegrates during the process.
The sheath of the E-coli is made up of lipopolysaccharides - as are all gram-negative bacteria - including the pathogen Lipid-A, which can be defined as an endotoxin. This would mean that in this association the released endotoxins are directly to blame as human pathogenic.
Subsequently, 272 patients suffering from PFS were subjected to stool diagnosis within two years (group A). 4185 stool examinations taken from the virology dept of the MHH served as a control group. 63 patients (group B) were selected from the survey group A and from these at least three stool samples were taken at an acute stage of their illness; these were used to confirm a closer association of the illness with evidence of coli-phages.
Group D, consisting of 30 patients from our survey who quite clearly did not suffer from PFS symptoms, was used as our control group. The results showed that specific coli phages were evident in the 225 of the 272 patients belonging to group A (=82.7%).
In the control group from the medical school there was evidence of phage infestation in merely 520 of the 4,185 persons tested (=12.4%). This result is highly significant. In the selected group B there was even clearer evidence of phages - 98.4% = 62 out of 63 persons. Results from the survey control group D lay by 13.3%, similar to control group C from the medical school (12.4%).
At the same time the department of immunology in the MHH analysed the endotoxins (LPS and lipid A) and the specific anti-bodies in the serum of the patients in group B for LPS and Lipid A in IGM and IGG form. Evidence of endotoxins was found directly in the serum of only 7 of the 63 patients (=11.1%), but specific anti-bodies against LPS and Lipid A were found in significantly greater numbers than in a healthy control group of blood donors.
The cause of the illness and its typical alternating course must be a consequence of a lysis of E coli infected with phages occurring in the bowels and induced by bacteriophages; endotoxins are subsequently released in the bowels and absorbed via the bowel wall into the blood system. Evidence of specific endotoxins and their anti-bodies in the serum constitute proof of this conjecture.
This means that the course of the illness begins with alimentary intake of T-bacteriophages (directly or in E coli bacteria in the lytic or lysogenic phase), followed by their colonisation and reproduction in the bowels and finally the lysis of the E coli, the release of endotoxins and their absorption. The idea that the release of endotoxins in the bowels is induced by phages and that Lipid A is absorbed into the blood system has not yet been recognised as a pathogenic process. 10 picogramm lipid A per ml serum is regarded as highly toxic!
According to this investigation primary fibromyalgic syndrome must primarily be regarded as an infection (bacteriophage infect of E. coli) and secondly as an intoxication (endotoxin) which enables us to understand the diverse occurrences of this illness with its chronically alternating course.
Theophil Hey, M.D. Braustraße 3 31675 Bückeburg www.praxishey.de
Arthritis Rheum 2002 Jun;46(6):1671-82 Related Articles, Books, LinkOut
Escherichia coli heat-labile enterotoxin B subunit prevents autoimmune arthritis through induction of regulatory CD4+ T cells.
Luross JA, Heaton T, Hirst TR, Day MJ, Williams NA.
Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK.
OBJECTIVE: The receptor-binding B subunit of Escherichia coli heat-labile enterotoxin (EtxB) is a highly stable, nontoxic protein that is capable of modulating immune responses. This study was conducted to determine whether mucosal administration of EtxB can block collagen-induced arthritis (CIA) and to investigate the mechanisms involved. METHODS: Clinical arthritis in DBA/1 mice was monitored following mucosal administration of EtxB on 4 occasions. The dependence of disease prevention on receptor binding by EtxB and the associated alterations to the immune response to type II collagen (CII) were assessed. Adoptive transfer experiments and lymph node cell cocultures were used to investigate the underlying mechanisms. RESULTS: Both intranasal and intragastric delivery of EtxB were effective in preventing CIA; a 1-microg dose of EtxB was protective after intranasal administration. A non-receptor-binding mutant of EtxB failed to prevent disease. Intranasal EtxB lowered both the incidence and severity of arthritis when given either at the time of disease induction or 25 days later. EtxB markedly reduced levels of anti-CII IgG2a antibodies and interferon-gamma (IFNgamma) production while not affecting levels of IgG1, interleukin-4 (IL-4), or IL-10. Disease protection could be transferred by CD4+ T cells from treated mice, an effect that was abrogated upon depletion of the CD25+ population. In addition, CD4+CD25+ T cells from treated mice were able to suppress anti-CII IFNgamma production by CII-primed lymph node cells. CONCLUSION: Mucosal administration of EtxB can be used to prevent or treat CIA. Modulation of the anti-CII immune response by EtxB is associated with a reduction in Th1 cell reactivity without a concomitant shift toward Th2. Instead, EtxB mediates its effects through enhancing the activity of a population of CD4+ regulatory T cells.
PMID: 12115200 [PubMed - indexed for MEDLINE]
Immunostimulant Patch Containing Heat-Labile Enterotoxin from Escherichia coli Enhances Immune Responses to Injected Influenza Virus Vaccine through Activation of Skin Dendritic Cells
Mimi Guebre-Xabier, Scott A. Hammond, Diane E. Epperson, Jianmei Yu, Larry Ellingsworth, and Gregory M. Glenn*
IOMAI Corporation, Gaithersburg, Maryland 20878
Received 29 October 2002/ Accepted 7 February 2003
Vaccine strategies, such as influenza virus vaccination of the elderly, are highly effective at preventing disease but provide protection for only the responding portion of the vaccinees. Adjuvants improve the magnitude and rates of responses, but their potency must be attenuated to minimize side effects. Topical delivery of strong adjuvants such as heat-labile enterotoxin from Escherichia coli (LT) induces potent immune responses. We hypothesized that LT delivered alone in an immunostimulating (LT-IS) patch placed on the skin at the site of injection could augment the immune response to injected vaccines. This was based on the observation that topically applied LT induces migration of activated antigen-presenting cells (APCs) from the skin to the proximal draining lymph node (DLN), and that APCs loaded with antigen by injection in the same anatomical region also migrate to the same DLN. We observed that when influenza virus vaccine is injected and an LT-IS patch is placed to target the same DLN, the influenza virus antibody response is enhanced. Similarly, influenza virus-specific T cells isolated from the lungs show increased levels of gamma interferon and interleukin-4 production. An LT-IS patch placed near an injected vaccine also leads to increased levels of hemagglutination inhibition titers, enhanced mucosal immunoglobulin A responses, and enhanced antigen presentation. Although the mechanisms by which an LT-IS patch exerts its enhancing effects need further study, the enhanced immune responses, ability to safely use potent adjuvants, and simplicity of LT-IS patch application address an important unmet need and provide a new immune enhancement strategy.
* Corresponding author. Mailing address: IOMAI Corporation, 20 Firstfield Rd., Suite 250, Gaithersburg, MD 20878. Phone: (301) 556-4500. Fax: (301) 556-4501. E-mail: email@example.com.
Journal of Virology, May 2003, p. 5218-5225, Vol. 77, No. 9
0022-538X/03/$08.00+0 DOI: 10.1128/JVI.77.9.5218-5225.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Clin Immunol 2003 May;107(2):116-21 Related Articles,>Links
Influenza vaccination and Guillain Barre syndrome small star, filled.
Geier MR, Geier DA, Zahalsky AC.
The Genetic Centers of America, 14 Redgate Court, 20905, Silver Spring, MD,
Acute and severe Guillain Barre Syndrome (GBS) cases reported following influenza vaccine to the Vaccine Adverse Events Reporting System (VAERS) database from 1991 through 1999 were examined. Endotoxin concentrations were measured using the Limulus amebocyte lysate assay in influenza vaccines. There were a total of 382 cases of GBS reported to the VAERS database following influenza vaccination (male/female ratio, 1.2). The median onset of GBS following influenza vaccine was 12 days (interquartile range, 7 days to 21 days). There was an increased risk of acute GBS (relative risk, 4.3; 95% confidence interval, 3.0 to 6.4) and severe GBS (relative risk, 8.5; 95% confidence interval, 3.7 to 18.9) in comparison to an adult tetanus-diphtheria (Td) vaccine control group. There were maximums in the incidence of GBS following influenza vaccine that occurred approximately every third year (1993, 1996, and 1998) and statistically significant variation in the incidence of GBS among different influenza manufacturers. Influenza vaccines contained from a 125- to a 1250-fold increase in endotoxin concentrations in comparison to an adult Td vaccine control and endotoxin concentrations varied up to 10-fold among different lots and manufacturers of influenza vaccine. The biologic mechanism for GBS following influenza vaccine may involve the synergistic effects of endotoxin and vaccine-induced autoimmunity. There were minimal potential reporting biases in the data reported to the VAERS database in this study. Patients should make an informed consent decision on whether to take this optional vaccine based upon its safety and efficacy and physicians should vigilantly report GBS following influenza vaccination to the VAERS in the United States so that continued evaluation of the safety of influenza vaccine may be undertaken.
PMID: 12763480 [PubMed - in process]
Transcutaneous Immunization with Tetanus Toxoid and Mutants of Escherichia coli Heat-Labile Enterotoxin as Adjuvants Elicits Strong Protective Antibody Responses
Rob Tierney,1 Anne-Sophie Beignon,2,a Rino Rappuoli,3 Sylviane Muller,2 Dorothea Sesardic,1 and Charalambos D. Partidos2
1Division of Bacteriology, National Institute for Biological Standards and Control, Potters Bar, United Kingdom; 2Unité propre de Recherche 9021, Institut de Biologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique, Strasbourg, France; 3Chiron SpA, Siena, Italy
Received 12 December 2002; accepted 27 March 2003; electronically published 11 August 2003.
In this study, the adjuvanticity of 2 nontoxic derivatives (LTK63 and LTR72) of heat-labile enterotoxin of Escherichia coli (LT) was evaluated and was compared with that of a cytosine phosphodiester-guanine (CpG) motif, after transcutaneous immunization with tetanus toxoid (TT). TT plus LTR72 elicited the strongest antibody responses, compared with those elicited by the other vaccines (TT, TT plus LTK63, TT plus CpG, and TT plus LTK63 plus CpG); it neutralized the toxin and conferred full protection after passive transfer in mice. Preexisting immunity to LT mutants did not adversely affect their adjuvant potency. Both LTK63 and LTR72 promoted the induction of IgG1 antibodies. In contrast, mice receiving either CpG motif alone or CpG motif plus LTK63 produced strong IgG2a anti-TT antibody responses. Overall, these findings demonstrate that mutants of enterotoxins with reduced toxicity are effective adjuvants for transcutaneous immunization.
Excerpt from http://www.medscape.com/viewarticle/461180_5
Dr. Shinefield: Our most recent results have turned up something fascinating about bacteremia in our population. The rate of bacteremia and of pneumococcal bacteremia is dropping, but we're finding Escherichia coli associated with pyelonephritis as the predominant cause of bacteremia in the toddler age group. These results are forcing us to rethink our approach to febrile toddlers.
Dr. Marcy: Considering the frequency of urinary tract infection in young children with fever, this may actually have been true for years. What we're seeing here is an unmasking of the E coli pyelonephritis because everything else seems to have disappeared. To get back to invasive disease, what do we do with the child who comes in with a febrile pneumonia? According to a study done last year by Isaacman and Burke, this is most likely to be pneumococcal pneumonia. In a situation like this, toxicity is the important factor. If the child is looking fairly well, despite a finding of rales or a suspicious chest radiograph, one would suspect an atypical pneumonia and proceed along those lines. If the child is toxic, I would not dramatically change my approach, whether or not they had received 1, 2, or 3 doses of the conjugate vaccine.
Common Genetic Damage Linked To Cancer And Aging
Two types of common DNA damage can create mutant proteins that can contribute to neurodegeneration, aging and cancer. Scientists at the Emory University School of Medicine used E. coli cells as a model system to study specific kinds of genetic damages that occur in all non-dividing cells undergoing transcription. They found two of the most frequently occurring spontaneous DNA damages -- both base substitution errors -- to which cells in all organisms are exposed on a daily basis cause transcriptional mutagenesis. TM occurs when cells with damaged DNA produce bad messages during transcription that lead to the creation of mutant proteins. "In some cases this miscoding could cause a cell to manufacture a mutant protein that controls cell division, which could take the cell from a non-growth state to a growth state and contribute to malignant transformation in the case of mammalian cells," said the lead author. The study was published in the Oct. 23 issue of Molecular Cell.
Bell palsy following intranasal vaccination
Bell palsy following intranasal vaccination
Results from a case-control study and a case-series analysis indicate a significantly increased risk of Bell palsy developing following intranasal immunization with a new vaccine. This inactivated influenza vaccine, composed of influenza antigens in a virosomal formulation with E. coli derived LT adjuvant, was licensed in Switzerland in October 2000.Following spontaneous reports of Bell palsy, the company decided not to market the vaccine during the following season. In general, the etiology and pathogenesis of Bell palsy remain inadequately understood. The greater risk of Bell palsy following immunization with this vaccine may be due to specific vaccine components such as LT toxin, influenza antigens or virosomes, or simply to use of the intranasal administration route. It is thus possible that such complications of vaccine administration may also apply to other nasal vaccines. GACVS therefore recommends that any novel vaccine for nasal administration should be tested on a sufficiently large number of subjects before licensing and submitted to active post-marketing surveillance studies. Since the average time to onset of Bell palsy following intranasal immunization with this new vaccine was as much as 60-90 days, GACVS recommends that the follow-up period in the context of clinical trials should be routinely extended to 3 months following administration of a new intranasal vaccine.
Intranasal Influenza Vaccine Strongly Linked to Bell's Palsy
NEW YORK (Reuters Health) Feb 25 - Immunization with an intranasal influenza vaccine used in Switzerland is strongly associated with the development of Bell's palsy, according to a new study. Nasalflu use was associated with a 84-fold increased risk of Bell's palsy, whereas parental flu vaccine use was not tied to an elevated risk.
The vaccine, which was marketed by Berna Biotech under the name Nasalflu, was first available for the 2000-2001 influenza season. From October 2000 to April 2001, the Swiss Drug Monitoring Center and other groups received 46 sentinel reports of vaccine recipients who had developed Bell's palsy.
Soon after, Berna Biotech stopped distributing the vaccine and invited Dr. Margot Mutsch, from the University of Zurich, and colleagues to investigate the possible link between Nasalflu use and Bell's palsy. In November 2001, the US Centers for Disease Control and Prevention joined the investigation.
In the new study, Dr. Mutsch's team identified 773 patients who were diagnosed with Bell's palsy in Switzerland during the period in which Nasalflu was in use. Of these patients, 250 were suitable for evaluation and were matched to 722 control subjects without the neurologic condition.
The intranasal vaccine had been used by 27.2% of Bell's palsy patients compared with just 1.1% of control subjects (p < 0.001), the researchers note. The findings are published in the February 26th issue of The New England Journal of Medicine
Concerned that their case-control study may have overestimated the risk seen with Nasalflu use, the researchers used other approaches to account for potential bias. However, even with the most conservative assumptions, Nasalflu users were 19 times more likely to develop Bell's palsy than controls. The highest risk of the disorder was seen between 31 and 60 days after vaccination.
In a related editorial, Dr. Robert B. Couch, from Baylor College of Medicine in Houston, discusses how the vaccine may have caused Bell's palsy and implications for future intranasal vaccines.
Nasalflu contained Escherichia coli enterotoxin as an adjuvant and it appears that this component, rather than the inactivated influenzavirus, was probably the risk-inducing factor, Dr. Couch notes. This component probably does not have a direct toxic effect but rather induces an autoimmune response or leads to reactivation of herpesvirus infection that causes the palsy.
"Identification of the cause of Bell's palsy associated with the use of the intranasal E. coli-adjuvant influenza vaccine and other vaccines is needed to facilitate further development of intranasal vaccines, including vaccines for influenza," Dr. Couch concludes.
N Engl J Med 2004;350:860,896-903.
Todar's Online Textbook of Bacteriology
Bordetella pertussis and Whooping Cough
© 2002 Kenneth Todar University of Wisconsin-Madison Department of Bacteriology
Whooping cough (pertussis) is caused by the bacterium Bordetella pertussis, B. pertussis is a very small Gram-negative aerobic coccobacillus that appears singly or in pairs. Its metabolism is respiratory, never fermentative, and taxonomically, Bordetella is placed among the "Gram-negative Aerobic Rods and Cocci" in Bergey's Manual. Bordetella is not assigned to any family. The bacteria are nutritionally fastidious and are usually cultivated on rich media supplemented with blood. They can be grown in synthetic medium, however, which contains buffer, salts, an amino acid energy source, and growth factors such as nicotinamide (for which there is a strict requirement). Even on blood agar the organism grows slowly and requires 3-6 days to form pinpoint colonies. Bordetella pertussis colonizes the cilia of the mammalian respiratory epithelium (Figure 1). Generally, it is thought that B. pertussis does not invade the tissues, but some recent work has shown the bacterium in alveolar macrophages. The bacterium is a pathogen for humans and possibly for higher primates, and no other reservoir is known. Whooping cough is a relatively mild disease in adults but has a significant mortality rate in infants. Until immunization was introduced in the 1930s, whooping cough was one of the most frequent and severe diseases of infants in the United States.
The disease pertussis has two stages. The first stage, colonization, is an upper respiratory disease with fever, malaise and coughing, which increases in intensity over about a 10-day period. During this stage the organism can be recovered in large numbers from pharyngeal cultures, and the severity and duration of the disease can be reduced by antimicrobial treatment. Adherence mechanisms of B. pertussis involve a "filamentous hemagglutinin" (FHA), which is a fimbrial-like structure on the bacterial surface, and cell-bound pertussis toxin (PTx). Short range effects of soluble toxins play a role as well in invasion during the colonization stage.
Figure 1. Colonization of tracheal epithelial cells by Bordetella pertussis
The second or toxemic stage of pertussis follows relatively nonspecific symptoms of the colonizaton stage. It begins gradually with prolonged and paroxysmal coughing that often ends in a characteristic inspiratory gasp (whoop). During the second stage, B. pertussis can rarely be recovered, and antimicrobial agents have no effect on the progress of the disease. This stage is mediated by a variety of soluble toxins.
Studies of B. pertussis and its adhesins have focused on cultured mammalian cells that lack most of the features of ciliated epithelial cells. However, some generalities have been drawn. The two most important colonization factors are the filamentous hemagglutinin (FHA) and the pertussis toxin (PTx). Filamentous hemagglutinin is a large (220 kDa) protein that forms filamentous structures on the cell surface. FHA binds to galactose residues on a sulfated glycolipid called sulfatide which is very common on the surface of ciliated cells. Mutations in the FHA structural gene reduce the ability of the organism to colonize, and antibodies against FHA provide protection against infection. However, it is unlikely that FHA is the only adhesin involved in colonization. The structural gene for FHA has been cloned and expressed in E. coli, raising the possibility of its production for use in a component vaccine.
One of the toxins of B. pertussis, the pertussis toxin (PTx), is also involved in adherence to the tracheal epithelium. Pertussis toxin is a 105 kDa protein composed of six subunits: S1, S2, S3, (2)
S4, and S5. The toxin is both secreted into the extracellular fluid and cell bound. Some components of the cell-bound toxin (S2 and S3) function as adhesins, and appear to bind the bacteria to host cells. S2 and S3 utilize different receptors on host cells. S2 binds specifically to a glycolipid called lactosylceramide, which is found primarily on the ciliated epithelial cells. S3 binds to a glycoprotein found mainly on phagocytic cells.
The S1 subunit of pertussis toxin is the A component with ADP ribosylating activity, and the function of S2 and S3 is presumed to be involved in binding the intact (extracellular) toxin to its target cell surface. Antibodies against PTx components prevent colonization of ciliated cells by the bacteria and provide effective protection against infection. Thus, pertussis toxin is clearly an important virulence factor in the initial colonization stage of the infection.
Since the S3 subunit of pertussis toxin is able to bind to the surface of phagocytes, and since FHA will attach to integrin CR3 on phagocyte surfaces (the receptor for complement C3b), it has been
speculated that the bacterium might bind preferentially to phagocytes in order to facilitate its own engulfment. The role of such self-initiated phagocytosis is not clear. Bacteria taken up by this abnormal route may avoid stimulating the oxidative burst that normally accompanies phagocytic uptake of bacterial cells which are opsonized by antibodies or complement C3b. Once inside of cells the bacteria might utilize other toxins (i.e. adenylate cyclase toxin) to compromise the bactericidal activities of phagocytes. In any case, there is some evidence that Bordetella pertussis can use this
mechanism to get into and to persist in phagocytes as an intracellular parasite. If B. pertussis is an intracellular parasite it would explain why immunity to pertussis correlates better with the presence of specific cytotoxic T cells than it does with the presence of antibodies to bacterial products.
B. pertussis produces at least two other types of adhesins, two types of fimbriae and a nonfimbrial surface protein called pertactin, but their role in adherence and pathogenesis is not well established.
Toxins Produced by B. pertussis
B. pertussis produces a variety of substances with toxic activity in the class of exotoxins and endotoxins. It secretes its own invasive adenylate cyclase which enters mammalian cells (Bacillus anthracis produces a similar enzyme, EF). This toxin acts locally to reduce phagocytic activity and probably helps the organism initiate infection. This toxin is a 45 kDa protein that may be cell-associated or released into the environment. Mutants of B. pertussis in the adenylate cyclase gene
have reduced virulence in mouse models. The organisms can still colonize but cannot produce the lethal disease. The adenylate cyclase toxin is a single polypeptide with an enzymatic domain (i.e., adenylate cyclase activity) and a binding domain that will attach to host cell surfaces. The adenylate cyclase was originally identified as a hemolysin because it will lyse red blood cells. In fact, it is responsible for hemolytic zones around colonies of Bordetella pertussis growing on blood agar. Probably it inserts into the erythrocyte membrane which causes hemolysis. An interesting feature of the adenylate cyclase toxin is that it is active only in the presence of a eukaryotic regulatory molecule called calmodulin, which up-regulates the activity of the eukaryotic adenylate cyclase. The adenylate cyclase toxin is only active in the eukaryotic cell since no similar regulatory molecule exists in procaryotes. Thus, the molecule seems to have evolved specifically to parasitize eukaryotic cells. Anthrax EF (edema factor) is also a calmodulin- dependent adenylate cyclase.
It produces a highly lethal toxin (formerly called dermonecrotic toxin) which causes inflammation and local necrosis adjacent to sites where B. pertussis is located. The lethal toxin is a 102 kDa protein composed of four subunits, two with a mw of 24kDa and two with mw of 30 kDa. It causes necrotic skin lesions when low doses are injected subcutaneosly in mice and is lethal in high doses. The role of the toxin in whooping cough is not known.
It produces a substance called the tracheal cytotoxin which is toxic for ciliated respiratory epithelium and which will stop the ciliated cells from beating. This substance is not a classic bacterial exotoxin since it is not composed of protein. The tracheal cytotoxin is a peptidoglycan fragment, which appears in the extracellular fluid where the bacteria are actively growing. The toxin kills ciliated cells and causes their extrusion from the mucosa. It also stimulates release of cytokine IL-1, and so causes fever.
It produces the pertussis toxin, PTx, a protein that mediates both the colonization and toxemic stages of the disease. PTx is a two component, A+B bacterial exotoxin. The A subunit (S1) is an ADP ribosyl transferase. The B component, composed of five polypeptide subunits (S2 through S5), binds to specific carbohydrates on cell surfaces. The role of PTx in invasion has already been discussed. PTx is transported from the site of growth of the Bordetella to various susceptible cells and tissues of the host. Following binding of the B component to host cells, the A subunit is inserted through the membrane and released into the cytoplasm in a mechanism of direct entry. The A subunit gains enzymatic activity and transfers the ADP ribosyl moiety of NAD to the membrane-bound regulatory protein Gi that normally inhibits the eukaryotic adenylate cyclase. The Gi protein is inactivated and cannot perform its normal function to inhibit adenylate cyclase. The conversion of ATP to cyclic AMP cannot be stopped and intracellular levels of cAMP increase. This has the effect to disrupt cellular function, and in the case of phagocytes, to decrease their phagocytic activities such as chemotaxis, engulfment, the oxidative burst, and bacteridcidal killing. Systemic effects of the toxin include lymphocytosis and alteration of hormonal activities that are regulated by cAMP, such as increased insulin production (resulting in hypoglycemia) and increased sensitivity to histamine (resulting in increased capillary permeability, hypotension and shock). PTx also affects the immune system in experimental animals. B cells and T cells that leave the lymphatics show an inability to return. This alters both AMI and CMI responses and may explain the high freqency of secondary infections that accompany pertussis (the most frequent secondary infections during whooping cough are pneumomia and otitis media).
Although the effects of the pertussis toxin are dependent on ADP ribosylation, it has been shown that mere binding of the B oligomer can elicit a response on the cell surface such as lymphocyte
mitogenicity, platelet activation, and production of insulin effects.
The pertussis toxin gene has been cloned and sequenced and the subunits expressed in E. coli. The toxin can be inactivated and converted to toxoid for use in component vaccines.
Figure 2. Comparison between cholera toxin and pertussis toxin (ptx) in their ability to interfere with the regulation of the eukaryotic adenylate cyclase complex.
Normal regulation of adenylate cyclase activity in mammalian cells (Adenylate cyclase (AC) is activated normally by a stimulatory regulatory protein (Gs) and guanosine triphosphate (GTP); however the activation is normally brief because an inhibitory regulatory protein (Gi) hydrolyzes the GTP.
Adenylate cyclase activated by cholera toxin (The cholera toxin A1 fragment catalyzes the attachment of ADP-Ribose (ADPR) to the regulatory protein Gs, forming Gs-ADPR from which GTP cannot be hydrolyzed. Since GTP hydrolysis is the event that inactivates adenylate cyclase (AC), the enzyme remains continually activated.
Adenylate cyclase activated by pertussis toxin (The pertussis A subunit transfers the ADP ribosyl moiety of NAD to the membrane- bound regulatory protein Gi that normally inhibits the eukaryotic
adenylate cyclase. The Gi protein is inactivated and cannot perform its normal function to inhibit adenylate cyclase. The conversion of ATP to cyclic AMP cannot be stopped.)
Lipopolysaccharide. As a Gram-negative bacterium Bordetella pertussis possesses lipopolysaccharide (endotoxin) in its outer membrane, but its LPS is unusual. It is heterogeneous, with two major forms differing in the phosphate content of the lipid moiety. The alternative form of Lipid A is designated Lipid X. The unfractionated material elicits the usual effects of LPS (i.e., induction of IL-1, activation of complement, fever, hypotension, etc.), but the distribution of those activities is different in the two forms of LPS. For example, Lipid X, but not Lipid A, is pyrogenic, and its O-side chain is a very powerful immune adjuvant. Furthermore, Bordetella LPS is more potent in the limulus assay than LPS from other Gram-negative bacteria, so it is not reliable to apply knowledge of the biological activity of LPS in the Enterobacteriaceae to the LPS of Bordetella. The role of this unusual LPS in the pathogenesis of whooping cough has not been investigated.
Regulation of Virulence Factors in B. pertussis
The production of virulence factors in B. pertussis is regulated in several different ways. Expression of virulence factors is regulated by the bvg operon. First, the organisms undergo an event called phase variation resulting in the loss of most virulence factors and some undefined outer membrane proteins. Phase variation has been shown to occur at a genetic frequency of 10-4 - 10-6 generations and results from a specific DNA frame shift that comes about after the insertion of a single nucleotide into the bvg (also known as vir) operon.
A similar process called phenotypic modulation, occurs in response to environmental signals such as temperature or chemical content, and is reversible. This is an adaptive process mediated by the products of the bvg operon, and is an example of a two-component environmental-sensing (regulatory) system used by other bacteria. The expression of these regulatory proteins is itself regulated by environmental signals, such that entry into a host might induce components required for survival and production of disease.
The Whooping Cough Vaccine
The development of the whooping cough vaccine in the 1950s has made whooping cough an uncommon disease in developed countries. In countries where the vaccine is not used whooping cough is an important cause of mortality in children, with an estimated 51,000,000 cases and 600,000 deaths annually. Historically, the whooping cough vaccine has been administered as a merthiolate-killed bacterial cell suspension which is part of the DTP vaccine (The P in DTP stands for Pertussis cells). Unfortunately, about 20% of the children that receive the whole cell vaccine experience mild side effects. About 0.1% of infants experience convulsions soon after receiving the vaccine and in a very small number of cases (1 in 150,000?) severe or irreversible brain damage may occur. In the absence of the disease in an immune population, parents have begun to wonder if the risk of vaccinating children outweighs the risk of the disease, and the value of the whole cell vaccine has been questioned.
Several new acellular vaccines have been developed from purified components of B. pertussis. Demonstration of the protective effects of anti-PTx and anti-FHA antibodies in the mouse model, focused vaccine production on combinations of inactivated pertussis toxin (toxoid) and filamentous hemagglutinin. Multicomponent acellular vaccines containing combinations of pertussis toxoid, filamentous hemagglutinin, pertactin, and the two types of fimbriae, are now being used in several countries including the U.S. The new vaccine, known as acellular pertussis has fewer side effects than the whole cell vaccine and is currently recommended for use under the conditions described below.
For decades, the pertussis vaccine has been given in combination with vaccines against diphtheria and tetanus. The combination is known as the DTP vaccine. Recently, infants have been able to receive a vaccine that combines the DTP vaccine with the vaccine against Haemophilus influenzae type b meningitis (Hib). This vaccine is called DTPH. The diphtheria-tetanus-pertussis vaccine using
acellular pertussis is known as DTaP. The diphtheria-tetanus-pertussis vaccination is given in five doses: at 2, 4, 6, 12-18 months and 4-6 years of age. Previously, DTaP had been recommended only for the fourth and fifth doses. Following FDA licensure of DTaP for infants, the Advisory Committee on Immunization Practices of the United States Public Health Service now recommends that DTaP be used for the first four doses and that DTaP still be used for the fourth and fifth doses for children who received DTP in their first three doses. The Committee is awaiting study results before making a
recommendation for the fifth dose for children who now will receive DTaP in their first four doses. The recommendation still permits the use of DTP and DTPH--the combination that includes the vaccine against Haemophilus influenzae type b meningitis.
For more information on whooping cough, see the CDC listings under Pertussis.
Study Results Hints at Future Intradermal Vaccine Against H. Influenza
NEW YORK (Reuters Health) Oct 01 - In infant rats, transcutaneous immunization with a glycoconjugate vaccine can produce protective antibody responses against Haemophilus influenza type B and diphtheria, according to a study in the September 15th issue of the Journal of Infectious Diseases
Dr. Fatme Mawas of the National Institute for Biological Standards and Control in Hertfordshire, United Kingdom, and colleagues there and elsewhere in Europe treated infant rats with transcutaneous administration of H. influenza type B cross-reacting material glycoconjugate vaccine, given together with cholera toxin or mutants of heat-labile Escherichia coli enterotoxin as adjuvants.
Transcutaneous immunization, the authors explain, "is a simple, noninvasive immunization procedure that targets the antigen-presenting cells residency in the epidermis." They applied the antigen solution directly onto the animals' bare skin. The conjugate vaccine "elicited high antibody responses to the capsular polysaccharide of H. influenza type B and to diphtheria toxin," according to the article.
Furthermore, the investigators report, passive transfer of diluted serum from immunized animals "completely protected infant rats from [H. influenza type B] bacteremia" and elicited strong neutralizing activity against diphtheria toxin. "Several challenges like ahead in terms of defining the stability and release kinetics of glycoconjugate vaccines incorporated into a patch and of defining the optimum immunogenic dose in humans," the authors admit. Nevertheless, they conclude, their results suggest "that this immunization strategy holds a lot of promise for future pediatric use."
J Infect Dis 2004;190:1177-1182.
Health Sciences Institute e-Alert
October 28, 2003
If you've suffered from it in the past, you know that the letters UTI can be three of the most painful in the English language. Just ask Delaney, a member who created an HSI Forum thread titled "Any natural cures for urinary tract infections?" She says, "I soooooo very much hate having to go to the doc and take an antibiotic. Is there another way?"
Good news, Delaney. There is another way. And it's often very effective. But before we get into that, I have some new research to tell you about that may reveal the reason why UTIs can be so irritating and hard to get rid of.
Lying in wait
In a recent issue of the journal Science, researchers at the Washington University (WU) School of Medicine in St. Louis explain that when Escherichia coli (E. coli) bacteria invade the bladder, they penetrate a protective coating of the superficial cells that line the bladder. Once the E. coli is established in the bladder lining, the stage is set for infection.
The WU team used mice to study bladder reaction to inoculations of E. coli. They found that after the bacteria began multiplying in the bladder lining, they formed pod structures that were protected by the cell coating. As the pods developed, they created special fibers that held them firmly in place below the coating. This type of cell organization is called biofilm. One of the authors of the study, Joseph J. Palermo, M.D., Ph. D., compared the pods beneath the coating to eggs protected by an egg carton.
The researchers found that after an infection was treated with antibiotics, pods that survived the treatment soon released bacteria to begin a new infection. Dr. Palermo and his colleagues believe that theirs' is the first study to reveal disease-causing biofilms that proliferate inside cells. They also believe that this explains why many bladder infections are often followed by subsequent infections after an initial successful treatment.
Location, location, location
Clearly, we're up against some very clever E. coli. So... what to do?
In response to Delaney's HSI Forum request for natural UTI treatments, several members offer methods that have worked for them. But among those treatments, one stands out. And a member named Les boils it down to a simple sentence: "D-mannose is the answer for UTI." Les is absolutely on the right track.
Many women know that when the first signs of a bladder infection appear, a few quarts of cranberry juice can sometimes head the problem off at the pass. According to Jonathan V. Wright, M.D., the ingredient in cranberry juice that makes it effective is the bacteria-fighting sugar Les recommends: D-mannose.
In the Health eTips e-letter "Inside Out" (9/11/03), Amanda Ross (Managing Editor of Dr. Wright's Nutrition & Healing newsletter) shared Dr. Wright's explanation about how D-mannose works. She wrote, "D-mannose... has the ability to detach E. coli from the walls of the bladder without upsetting the balance of the friendly bacteria necessary for good health. After being loosened from bladder walls, the bacteria are rinsed away by normal urination. The E. coli aren't killed; they're simply relocated - 'from the inside to the outside' - and the infection is gone."
Cranberry... without the cocktail
But before you run out to stock up on cranberry juice, you should know that Dr. Wright says there's not really enough D-mannose in the juice to be significantly effective. And in her article, Amanda adds that the extra sugar that comes with most brands of cranberry juice just creates other unneeded problems.
Fortunately, D-mannose is available from compounding pharmacies and many natural food stores. Dr. Wright says that almost any bladder infection caused by E. coli can be eliminated with 1/2 to 1 teaspoonful of D-mannose, dissolved in water and taken every 2 to 3 hours. And there's no need to worry about the sugar aspect of D-mannose; it's a simple sugar, so very little of it is metabolized by the body. Large doses are washed away in the urine, and the amounts not excreted into the urine are so small that they do not affect blood sugar levels - even in diabetics.
And for a personal testimonial about the effectiveness of D- mannose we'll go back to the HSI Forum thread where a member named Rosie shared this experience: "I finally decided to try the D-Mannose that Dr. Wright raves about for UTI's. I took one half teaspoon a day, less than what the directions say to use. In two days the UTI was gone, but I continued the D-Mannose a few more days."
Judging from the Washington University study, Rosie had the right idea to continue the treatment for a few days to continue fighting any remaining bacteria that the E. coli pods might try to reintroduce in the bladder.
Brain Res Dev Brain Res. 2005 May 5; [Epub ahead of print]
Suppression of glial activation is involved in the protection of IL-10 on maternal E. coli induced neonatal white matter injury.
Pang Y, Rodts-Palenik S, Cai Z, Bennett WA, Rhodes PG.
Department of Pediatrics, University of Mississippi Med. Ctr., Jackson, MS 39216-4505, USA.
White matter damage (WMD) is an important cause of disability including cerebral palsy in preterm, low birth-weight infants. Maternal infection is now recognized as one of the risk factors for WMD. Previously we reported that intrauterine inoculation of Escherichia coli to pregnant rats resulted in WMD in offspring and interleukin-10 (IL-10) was protective against this damage. The objective of this study was to elucidate the mechanism involved in the protective effect of IL-10 against neonatal WMD. We found that E. coli treatment in dams resulted in significant apoptosis in periventricular white matter of rat pups on postnatal day 0 (P0). On P8, a remarkable increase in ED-1 immunostaining (indicating either microglial activation or macrophage infiltration) was detected in brains of pups in the E. coli-treated group. Astrogliosis was also noticed in brain white matter of pups in the E. coli-treated group. In addition to the strong activation of microglia and astrocytes, oligodendrocytes (OLs) were significantly reduced in periventricular areas in the brains of pups from the E. coli-treated group. Later, on P15, hypomyelination was also noticed in rat brains from the E. coli-treated group, using myelin basic protein (MBP) immunostaining. Treatment with IL-10 after E. coli inoculation significantly reduced TUNEL staining and caspase-3 activation, and partially restored the impaired immunostaining markers for immature and mature OLs, such as CNPase, O4, adenomatous polyposis coli (APC) and MBP. These results indicate that the protective effect of IL-10 against brain WMD is linked with suppression of microglial activation/macrophage infiltration, as shown by significantly reduced ED-1+ cells in the white matter.
Alzheimer's-Causing Amyloid And Bacteria Trigger Same Immune Response In The Brain 24 Jul 2009
In a new study published today in the July issue of the journal Cell Host & Microbe, UC Davis researchers report that both amyloid plaques found in the brains of Alzheimer's patients and structures made by some gut bacteria likely elicit the same response by human immune cells.
"Alzheimer's disease may be a case of mistaken identity," said Andreas Bäumler, a professor of microbiology and medical immunology. Bäumler and his colleagues showed that the immune systems of mice injected with E. coli and salmonella are triggered by curli fibrils, fiber-like structures consisting of curli proteins that allow bacteria to stick to host tissue and to each other and form colonies.
Curli fibrils are morphologically identical to amyloid fibrils found in Alzheimer's plaques. When they presented human cells with the two kinds of fibrils, they saw the same immune response - even though the two have nothing in common in their amino acid sequences.
"Our results suggest that it's the structure of these protein aggregates that matter and that, to the innate immune system, Alzheimer's plaques may look like colonies of bacteria. This would result in the chronic inflammation we see in Alzheimer's disease that damages neurons," Bäumler explained.
Amyloid plaques are the sticky buildup of proteins that accumulate outside nerve cells. They are characteristic of several illnesses, including Alzheimer's disease, Huntington's disease, type 2 diabetes, secondary amyloidosis and prion diseases, like Creutzfeldt-Jakob (the human form of mad cow disease). These diseases all involve marked inflammation at the sites of amyloid deposition, resulting in tissue injury.
The protein forming plaques in Alzheimer's patients is normally soluble. When the protein folds improperly, it forms amyloid deposits that are associated with brain inflammation. Until now, scientists have not been able to identify what causes this destructive, chronic inflammation.
Bäumler and his colleagues did not expect to be studying Alzheimer's disease. They were studying inflammation of the gut caused by bacteria when they discovered that the innate immune system was being triggered by a structural feature of bacterial amyloids and not by the amino acids that make up the proteins in the biofilms.
"When we destroyed the ability of the proteins to aggregate, we no longer saw the same immune response," Bäumler said.
When the researchers figured out the amyloid structure was responsible for triggering the immune system, they decided to see whether the same immune response was being triggered by structurally identical amyloids associated with human disease. They chose to study the synthetic form of the proteins that make up Alzheimer's plaques because the disease has been widely studied and a lot is known about the biochemistry of these plaques.
In the current study, Bäumler and his team found that, in mice, the immune response to curli fibrils is controlled by a protein called TLR-2 (Toll-like receptor 2). TLR-2 is expressed on the surface of certain cells that recognize foreign substances and passes on appropriate signals to the cells of the immune system.
Next, the team used cultures of human immune system cells to see if synthetic versions of the major curli fibril protein (CsgA) and the major protein found in amyloid plaques, (beta-amyloid) would trigger the TLR-2 response. They used cultures of macrophage cells, immune cells that engulf and digest cellular debris and pathogens. They also used microglia, which are essentially macrophages of the brain.
The team found that the mechanism involving TLR-2 was triggered by both CsgA and beta-amyloid but only when these proteins were allowed to aggregate into amyloids.
"The CsgA peptide and beta-amyloid don't have anything genetically in common. The similarity is only in the structure of the amyloids they form. Our study indicates that there is some structural feature of amyloids that triggers the innate immune system," Bäumler said.
The current findings are only the first step in what may ultimately be a drug treatment for amyloid diseases. If researchers can find a drug that inhibits the TLR-2-dependent immune response, Bäumler said, they could potentially slow down the progression of amyloid diseases, including Alzheimer's.
"You have to know what causes the diseases in order to find the right drug target. Now we know what's causing the chronic inflammation associated with amyloids. It's a start, though we're still a long from finding a treatment," he said.
Additional authors of the study include UC Davis' Cagla Tükel, R. Paul Wilson, Jessalyn Nishimori and Milad Pezeshki; and Brett Chromy of Lawrence Livermore National Laboratory.
The study was funded by Public Health Service grants to Bäumler and a Scientist Development Grant from the American Heart Association to Çagla Tükel.
UC Davis School of Medicine
Article URL: http://www.medicalnewstoday.com/articles/158628.php