carbonchio

Master in Medicina NBC - Master in Medicina NBC - 20112011

Carbonchio

Prof. Pasquale Urbano

Collegamento al Consensus e alle linee guida europee

P. Urbano per il Master in Medicina NBC – 2011

CarbonchioNon è una cosa nuova, e in italiano si è sempre chiamato così

È sbagliato chiamarlo ‘antrace’, come fanno i media e come, deplorevolmente, sta entrando nell’usoI vecchi testi lo trattano estesamente


Le microfotografie originali di Robert Koch,

sul bacillo del carbonchio


Vaccino anticarbonchioso

Storicamente, il carbonchio è la prima malattia per prevenire la quale fu dimostrato efficace un vaccino battericoLe colture attenuate ottenute da Pasteur dimostrarono la loro efficacia immunizzante nel famoso esperimento di Pouilly Le Fort nel 1881.


Vaccino anticarbonchiosoIl vaccino disponibile per uso umano è poco efficacePaxVax Inc. of San Diego will develop and test two formulations of an anthrax vaccine. Both formulations contain a gene known as rPA, which codes for a protein that protected animals from anthrax in previous studies. The gene will be inserted into a carrier virus or vector called adenovirus 4 and the vaccine delivered orally via a capsule. The adenovirus 4 vector is a modified live-virus vaccine that has been studied for more than 40 years and has a good safety profile in humans. Pending review and approval of the Investigational New Drug Application by the Food and Drug Administration, a Phase 1 clinical trial will be conducted to select one of the two formulations for further development. NIAID has made an initial award of $3.8 million to PaxVax. If milestones are met, the total award could amount to $23.8 million over the next nine years.http://www.dddmag.com/news-NIH-Funding-Bioterror-Vaccines-10810.aspx


Vaccino anticarbonchiosoEmergent Biosolutions of Rockville, Md., also will develop and test an anthrax vaccine. The candidate vaccine consists of Emergent's licensed anthrax vaccine (BioThrax) combined with an adjuvant. The investigational vaccine was tested in a Phase 1 clinical trial prior to the award of this contract. This new award will fund manufacturing, stability studies, assay validation and clinical trial preparation of the vaccine product. After those activities are completed, the vaccine may begin Phase 2 clinical testing. Emergent has received an initial award of $9.1 million, with the potential for total funding of up to $28.7 million over the next four years if clinical milestones are met.http://www.dddmag.com/news-NIH-Funding-Bioterror-Vaccines-10810.aspx


Diagnosi sulle carogne

Termoprecipitazione di Ascoli [1911]

Estratti a caldo, filtrati, vengono stratificati in una provetta sottile contenente siero anticarbonchiosoLa formazione di un anello di precipitato è diagnostica

Importante presidio di profilassi generale


Carbonchio in Italia - 2011At the moment there are 39 suspected animals in Campania region (9

confirmed); and 14 in Basilicata.

V. nota

P. Urbano per il Master in Medicina NBC – 2011Carbonchio


CarbonchioAgente: Bacillus anthracisSpore e tossineForme cutanee, inalatorie****, intestinali

**Sospettare il bioterrorismo**Sospettare il bioterrorismo


Forma vegetativa di B. anthracis, da coltura. Gram


Bacillus anthracis, SEM colorata


La virulenza dipende dalla capsula, governata dal plasmide pXO2

La patogenicità dipende da LF e EF, che si associano a PA a formare le esotossine, governate dal plasmide pXO1

Genetica


CapsulaPolimero di acido D-gamma-glutammico, formato da CapD, una transpeptidasi codificata nel plasmide pXO2Costituisce il principale fattore di virulenza, non trovando recettori sui macrofagi


Tossine del Carbonchio

I componenti della tossina sono tre:• OF [Oedema Factor]• LF [Lethal Factor]• PA [Protective Antigen]

Agiscono di concerto; isolatamente, non sono tossici


B. anthracis, geni per le

tossine


PA del CarbonchioE’ la componente deputata a riconoscere il recettore, l’ATR [Anthrax Toxin Receptor], una proteina di membrana di tipo I, che ha un dominio extracellulare A del Fattore di von Willebrand.Legata al recettore, PA viene clivato in una parte che si perde e in un frammento carbossiterminale che eptamerizza, acquisendo affinità per OF e/o LF, che possono così essere endocitati e agire nel citosol.


Intossicazione da B. anthracis


Fattore Edemigeno (EF) del carbonchio

• 89 kDa

Gene su plasmide pXO1adenilato ciclasi calcio- e calmodulina-dipendente, che porta alla formazione di edema e all’inibizione delle funzioni dei polimorfonucleati


Fattore Letale (LF) del Carbonchio

•90 kDa, gene sul plasmide pXO1

•LF è una zinco-endoproteasi che taglia l’aminotermine delle protein-chinasi mitogeno attivate 1 e 2 (MAPK K1 e K2), inattivandole, e così bloccando la trasduzione del segnale di attivazione

Nei macrofagi: attivazione del burst ossidativo

rilascio di ossigeno reattivo induzione di citochine proinfiammatorie, quali TNF e IL-1ß shock e morte


Spora del carbonchio e macrofago


Carbonchio Cutaneo

Incubazione 1-12 gg

Papula -> vescicola o ulcera -> escara centrale nera con alone rosso vivo, per diversi giorni

Diagnosi: Batterioscopia (Gram) e coltura da vescicole, base dell’ulcera, sotto il margine dell’escara

Buona risposta al trattamento


Carbonchio Cutaneo


Carbonchio cutaneo

Realistico moulage in cera, nel museo al Chiostro del Maglio


Epidemia di carbonchio

cutaneo

Nella prima guerra mondiale l’esercito inglese fu colpita da una vasta epidemia di carbonchio cutaneo, a localizzazione sul viso e allo scalpo, per l’impiego di spazzole e pennelli da barba prodotti in India con setole contaminate da spore carbonchiose


Carbonchio da inoculazione•È in corso un’epidemia fra gli eroinomani, in Scozia, UK e in Germania

•Contaminazione con spore di eroina proveniente dall’Afganistan•35 casi, 11 morti, al 22 aprile 2010


Carbonchio da inoculazione•È stata ventilata e discussa una possibile

causa dolosa, bioterroristica, della

contaminazione con spore carbonchiose

dell’eroina grezza che sta causando

l’epidemia in Scozia e Inghilterra

•È considerata plausibile, ma improbabile

e praticamente non provabile

•Vedi nota


Carbonchio da inoculazioneSu Lancet, Vol 375 April 17, 2010, p. 1345 è presentato il quadro clinico di questa nuova forma di carbonchio.Estratto in: http://download.thelancet.com/pdfs/journals/lancet/PIIS0140673610605739.pdf?id=3d35b1b5aa0ec416:-47766f9:12824058b61:68391271920107987


PROCEDURE OPERATIVE PER LA

GESTIONE DI MATERIALE

POTENZIALMENTE CONTAMINATO DA

Bacillus anthracis


Resistenza sporeLe spore resistono per tempi lunghissimi, indefiniti, agli agenti naturali di disinfezione

Spore carbonchiose reperite negli intonaci dei più vecchi tronchi della metropolitana londineseSpore ancora attive dopo 48 anni, nell’isola di Gruinard contaminata per provare la dispersione con testate di artiglieriaRischio per scavi attraverso sito sepolcrale del 16° secolo vedi nota


BBC, 18 marzo 2010


Gruinard IslandNel 1942, utilizzata per esperimenti di guerra biologica con spore carbonchiose.Scelta per la lontananza da luoghi molto popolati; confiscata ai pochi abitanti, poi risarciti, l’isola è servita per provare le modalità di dispersione di spore caricate su ordigni esplosivi.Il successo delle prove è stato dimostrato dalla moria del bestiame ripetutamente portato a pascolare sull’isola. [Interdizione di area]L’isola risultava ancora contaminata negli anni ‘80.Nell’86 grossa operazione dei decontaminazione

280 tonnellate di formaldeide in acqua di mare spruzzata sui 196 ettari di superficie buon esito e fine della quarantena


Operation Dark Harvest

In 1981 British newspapers began receiving messages with the heading "Operation Dark Harvest" which demanded that the government decontaminate the island, and reported that a "team of microbiologists from two universities" had landed on the island with the aid of local people and collected 300 lbs of soil. The group threatened to leave samples of the soil "at appropriate points that will ensure the rapid loss of indifference of the government and the equally rapid education of the general public". The same day a sealed package of soil was left outside the military research facility at Porton Down; tests revealed that it contained anthrax bacilli. A few days later another sealed package of soil was left in Blackpool, where the ruling Conservative Party was holding its annual conference. The soil did not contain anthrax, but officials said that the soil was similar to that found on the island.[10]

Bioterrorismo bello e buono !Bioterrorismo bello e buono !Bioterrorismo bello e buono !Bioterrorismo bello e buono !


Decontaminazione - cautele

Per agire in zone contaminate da agenti biologici occorrono cautele particolari

Presidi personali di protezioneVestizioneLavaggioSvestizione


Carbonchio da inalazione

La forma clinica che si teme a seguito di un attacco

massiccio, per aerosolizzazione delle spore

carbonchiose, è il


Nature. 2001 Nov 8;414(6860):225-9. Identification of the cellular receptor for anthrax

toxin.Bradley KA, Mogridge J, Mourez M, Collier RJ, Young JA.

The tripartite toxin secreted by Bacillus anthracis, the causative agent of anthrax, helps the bacterium evade the immune system and can kill the host during a systemic infection. Two components of the toxin enzymatically modify substrates within the cytosol of mammalian cells: oedema factor (OF) is an adenylate cyclase that impairs host defences through a variety of mechanisms including inhibiting phagocytosis; lethal factor (LF) is a zinc-dependent protease that cleaves mitogen-activated protein kinase kinase and causes lysis of macrophages. Protective antigen (PA), the third component, binds to a cellular receptor and mediates delivery of the enzymatic components to the cytosol. Here we describe the cloning of the human PA receptor using a genetic complementation approach. The receptor, termed ATR (anthrax toxin receptor), is a type I membrane protein with an extracellular von Willebrand factor A domain that binds directly to PA. In addition, a soluble version of this domain can protect cells from the action of the toxin.


Sensor To Detect Agents Used In Biological WarfareResearchers from the University at Buffalo are developing a handheld sensor that can detect the presence of toxins potentially used as agents in biological warfare.

The proposed sensor, which will utilize optical-detection and chemical-sensing technologies, could be used in urban, military, industrial and even home environments,

The sensor will be composed of three components -- a LED (light emitting diode), a xerogel-based sensor array and a CMOS (complementary metal-oxide semiconductor) detector, commonly used in miniature digital cameras.


The test uses a fluorescent 'substrate' to indicate the activity of lethal factor protease. Two fluorescent probes are attached to a synthesized target—a small peptide that looks like proteins cleaved by lethal factor in human cells. The cutting of the target molecule produces a fluorescent glow.Proceedings of the National Academy of Sciences


Nature. 2001 Nov 8;414(6860):229-33.Crystal structure of the anthrax lethal factor.

Pannifer AD, Wong TY, Schwarzenbacher R, Renatus M, Petosa C, Bienkowska J, Lacy DB, Collier RJ, Park S, Leppla SH, Hanna P, Liddington RC.

Lethal factor (LF) is critical in the pathogenesis of anthrax. It is a highly specific protease that cleaves members of the mitogen-activated protein kinase kinase (MAPKK) family near to their amino termini, leading to the inhibition of one or more signalling pathways. LF comprises four domains: domain I binds the membrane-translocating component of anthrax toxin, the protective antigen (PA); domains II, III and IV together create a long deep groove that holds the 16-residue N-terminal tail of MAPKK-2 before cleavage. Domain II resembles the ADP-ribosylating toxin from Bacillus cereus, but the active site has been mutated and recruited to augment substrate recognition. Domain III is inserted into domain II, and seems to have arisen from a repeated duplication of a structural element of domain II. Domain IV is distantly related to the zinc metalloprotease family, and contains the catalytic centre; it also resembles domain I. The structure thus reveals a protein that has evolved through a process of gene duplication, mutation and fusion, into an enzyme with high and unusual specificity.


Avanzamento conoscenze

Recenti indagini hanno

chiarito le tappe della

sporogenesi e identificato

le circa 750 proteine che si

ritrovano nelle spore

•L’Istituto per la Ricerca Genomica (TIGR) di Rockville, Md, ha sequenziato l’intero genoma del B. anthracis, e ha poi creato una micromatrice che lo rappresenta tutto, per indagare l’attivazione sequenziale dei geni durante la sporogenesi.

Spora di B anthracis, 92,000x


Avanzamento delle conoscenze

To detect the presence of the toxins, the researchers will produce sensors called Protein Imprinted Xerogel with Integrated Emission Sites (PIXIES). Within the PIXIES, a tiny fluorescent dye molecule is placed within the xerogel's imprint sight. The PIXIES then are placed atop the LED, which is used to stimulate the fluorescent dye to emit light.The fluorescent molecule is sensitive to the presence of other molecules in its immediate environment. Thus, when the target toxin is recognized by the PIXIES, the fluorescent molecule will change its light intensity


"MASTER SWITCH." Another microbial enzyme, known as DNA adenine methylase (DAM), is the target of Remedyne Corp., a startup founded by scientists at the University of California, Santa Barbara. The researchers discovered that DAM is a "master switch" that controls more than 20 genes that are critical to many bacteria's ability to infect their hosts. When they engineered bacteria with a defective form of DAM, the virulent bugs turned into pussycats: They survived -- but they couldn't cause disease.


Darrell Galloway, a microbiologist at Ohio State University, and colleagues at the National Institutes of Health and the Naval Medical Research Center, isolated the DNA from three genes that work in concert to create the deadly cascade of toxins secreted by anthrax bacteria. Using viral carriers, called plasmids, they incorporated these genetic snippets into the cells of healthy mice.

When the treated mice were injected with five times the lethal dose of the anthrax bacterial toxin, those that received the plasmid injections were immune. Every animal in the control group died within hours. Moreover, the Ohio State team, in collaboration with scientists from researcher Battelle Group, have shown that the vaccine protects against aerosol-delivered anthrax -- the most deadly form -- more than a year later.


Antibodies are the key to a third strategy being developed by EluSys that sops up microorganisms or toxins and ferries them out of the body. The tricky molecules, which the company calls Heteropolymer (HP) technology, consist of two antibodies that are chemically bound together. One attaches the molecule to a red blood cell, the other is designed to bind to the biowar agent, such as the anthrax toxin. The immobilized biowar agents then get a ride on the red blood cells to the liver, where they are destroyed. "It's like two-sided sticky tape," explains George L. Spitalny, the company's vice-president for R&D.


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Figure. Number of environmental specimens submitted to the Illinois Department of Public Health Division of Laboratories for Bacillus anthracis testing each week from October 8 through December 30, 2001 and number of human cases occurring on the East Coast and reported each week in the news media.

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Falsi allarmi dopo veri casiEID, Volume 9, Number 4, April 2003


CarbonchioAnthrax FAQ, 2005Anthrax as a Biological Weapon, 2002: Updated Recommendations for Management[JAMA 287(17),2002]Anthrax as a Biological Weapon: Medical & Public Health Management (1999)[JAMA 281(18),1999]Anthrax Fact Sheet, 2002Anthrax Fact Sheet, 1999Anthrax BibliographyConsidering Anthrax Vaccine for Post- Exposure ProphylaxisSlides Presented at Meeting on Anthrax Vaccine, National Academy of Sciences, December 15, 2001Clinical Anthrax: Primer for PhysiciansCase Studies of Bioterrorism-Related Inhalational AnthraxFAQ: Environmental Detection of Anthrax


ROC per CarbonchioEID, Volume 10, Number 1, January 2004

Master in Medicina NBC - Master in Medicina NBC - 20112011

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