CASE CLOSED … what really happened in the 2001 anthrax attacks?

* FBI anthrax investigation … statements from DOJ & FBI regarding the FBI’s anthrax science

Posted by DXer on January 10, 2010

thanks to Anonymous Scientist for these materials …

click to open …

DOJ Science Anthrax Press Conference – 8-18-08

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FBI Assistant Director Dr. Vahid Majidi – science of the anthrax investigation – 8-18-08

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FBI Assistant Director Hassell – statement to NAS 7-30-09

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Fraser-Liggett et al – The Genomics Behind the Amerithrax Investigation ***

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*** Anonymous Scientist comments … Slide 31 of the Fraser-Liggett presentation shows a picture of the Fermentor at Dugway and a batch of flasks at Detrick. It states that 12 X 10 liter runs were performed at Dugway and 22 flask preparations at Detrick to finally concentrate down to a 1 liter flask of RMR-1029.

RMR-1029 basically consisted of 30 grams of spores in 1 liter of water. Obviously enormous resources were used to produce it.  If we assume that at least 10 grams of spores were needed for the letters (you would lose powder at every step) – it shows the impossibility of this being done by Ivins over a few evenings alone.

Fraser-Liggett slide #31

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CASE CLOSED is a novel which answers the question … Why did the FBI fail to solve the 2001 anthrax case?

Here’s the (fictional) DIA Director giving the charge to his team re-investigating the FBI anthrax investigation …

“Those FBI bastards hounded a Defense Department employee until he committed suicide, if it was suicide. After seven years the FBI hasn’t come close to making a case that could convict the lowest grade criminal, let alone an internationally respected scientist. And they think they can say ‘case closed’ and sweep their incompetent investigation under the rug?”

“I’ve already spoken to Secretary Morgan,” General Drysdale continued. “The Secretary agrees that the Defense Department is taking an unwarranted hit from the FBI, and we don’t know why. At my request, the Secretary has authorized us to find out what really happened.

“You’re the team I’ve selected. You’re authorized to go where you need to go, ask what you need to know. You’ll have whatever resources are necessary.

Click here to buy CASE CLOSED by Lew Weinstein

in paperback or kindle

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9 Responses to “* FBI anthrax investigation … statements from DOJ & FBI regarding the FBI’s anthrax science”

  1. DXer said

    Vahid Majidi thinks the August 17, 2008 press conference on the forensic science was a big mistake. I agree.

    In that press conference, he says:

    “Finally, I am asking you to understand that this is the first step toward broader dissemination of the scientific information surrounding this case. Additional information will be available through peer reviewed publications and I ask that you to please respect the integrity of this process.”

    Huh?

    What projects were they, Vahid? Do you mean the excellent report on the genetic distinctiveness of the subtilis? That report admittedly is helpful in understanding the significance of your decision not to swab all the suspect labs for the genetically distinctive subtilis.

    What other peer-reviewed reports were published that supported your Ivins Theory? None?

    But by all means, GAO should obtain any reports submitted for publication that were rejected so as to have benefit of the material.

  2. DXer said

    (6)
    (b)01/18/2007 01:25
    PM Subject
    Question for you

    Hi,
    I have a question for you. Did we ever use a fermentor to grow anthracis, either to make toxin
    or spores? I know that a fermentor was on my handreceipt in 1992, but by 1993 it was off. I don’t
    recall our ever using one then, but I could be wrong. Thanks!
    Bruce Ivins

  3. DXer said

    Claire Fraser-Liggett, professor at the University of Maryland School of Medicine and director of the University of Maryland Institute for Genome Sciences, asked,

    “What would have happened in this investigation had Dr. Hatfill not been so forceful in his response to being named a person of interest. What if he, instead of fighting back, had committed suicide because of the pressure? Would that have been the end of the investigation?”
    ***
    “I have complete confidence in the accuracy of our data,” Fraser-Liggett says, but she emphasizes that it fails to show Ivins is guilty.

  4. DXer said

    Tarek Hamouda the scientist who in presentations, articles, and patents says he was supplied virulent Ames strain by Bruce Ivins in connection with DARPA work using a lyophilizer, obtained his PhD in 1996 from Cairo Medical, Ayman Zawahiri’s alma mater. The professor of pharmacology there was a woman named Heba Al-Zawahiri. She is Ayman’s sister. Her research has included issues relating to anti-inflammation and antimicrobials. Ayman Zawahiri’s uncle was the Dean of Cairo’s medical school. Ayman Zawahiri’s father, who passed away in 1995, was also a Professor of Pharmacy.

    Ayman Zawahiri was the medical doctor who Egyptian intelligence says made 15 attempts over 10 years to recruit a scientist who could assist in the use of anthrax against US targets. He understood the koran and hadiths to command that the jihadists use the weapons of his enemy. A memo he wrote to fellow Egyptian Atef, a former Cairo policeman, show that he calculated that the most expeditious means of developing anthrax as a weapon was to recruit specialists using the cover of universities and charities.

    Dr. Hamouda — provided virulent Ames by Bruce Ivins — likely would have keen insights into the upset that Professor Heba A-Zawahiri family felt about the rendering of Heba’s two brothers, Mohammad and Hussain. He had gone to medical school and graduated in December 1982, at the height of the frenzy surrounding the aftermath of Sadat’s assassination. Heba had been under the strain for years of having had her brothers Mohammad and Hussain rendered and detained by Egyptian authorities. She perceives (not unreasonably) that they had been physically mistreated. Under the “Leahy Law,” appropriations continued to Egyptian security units notwithstanding such mistreatment. Islamist publications show that this was noted shortly before the anthrax mailings.

    When I first contacted them a long time ago, Dr. Hamouda and his colleagues — although they received millions from DARPA — refused to even provide a copy of his presentations to ASM describing his research involving Bruce Ivins or the presentation made by Michael Hayes to ICAAC. Michael this past week politely told me when I called to ask for a copy of the presentation that “You don’t want to speak to me and I don’t want to speak to you.”

    This is why I get so angry when FOIA officers don’t comply with the law and when the scientists feeding at the public trough get arrogant. It is not right to take a lot of money from taxpayers — such as the $890,000 paid the staff of the National Academy of Sciences or the millions paid Sandia — and then not comply with the Government in the Sunshine Act. If Dr. Baker and University of Michigan does not want to comply with FOIA, they should refund the money provided by DARPA. If virulent Ames was provided the former doctoral student associated with Ayman Zawahiri’s sister, and the government funded his later research with Bruce Ivins, then as a condition of receipt of such funds there should be compliance with FOIA.

    The torment and worry that Pharmacology Professor Heba Zawahiri felt was understandable. It is the same stress felt by Fowzia Siddiqui, whose sister, according to her psychiatrist, says she was tasked to study germ weapons under a fatwa. She had been a professor at John Hopkins before returning to Pakistan to advocate for her sister.

    Human Rights Watch explained in 2005 Professor Heba Al-Zawahiri’s travails:

    “At the time he was rendered from UAE to Cairo in March or April 1999, Muhammad al-Zawahiri had not set foot on Egyptian soil for a quarter-century.”

    [This was when the blind sheik’s lawyer Al-Zayat announced that Ayman Zawahiri was going to use anthrax against US targets in retaliation for the rendering of senior Movement leaders.]

    Human Rights Watch continues:

    “After graduating from the faculty of engineering at Cairo University, Muhammad, the brother of senior al-Qaeda leader Ayman al-Zawahiri, left Egypt to work for various construction firms in Saudi Arabia. In 1981, his name was on the list of defendants in the mass trial of alleged conspirators in the assassination of President Sadat.”

    [The second mailing was on the data of Sadat’s assassination; the first mailing was on the date that the Camp David agreement was approved. Ayman Zawahiri explains the significance of both dates in his October 7, 2001 book.]

    “Despite his acquittal, Muhammad was still wary of returning to Egypt after that,” his uncle said. “He was worried that they would accuse him again.”

    [In a footnote, Human Watch Rights note that Al-Zayat and Ayman Zawahiri have indicated that Muhammad had been in the same militant cell as Ayman.]

    “After an Egyptian imam of a mosque in Jeddah was arrested by Saudi authorities and returned to Cairo in the early 1990s, Muhammad began to prepare for his own departure.”
    He took his family first to Yemen, and then to Sudan, where he was reunited with Ayman. Both Muhammad and Ayman were forced to leave Sudan in 1995; Ayman returned to Afghanistan, and Muhammad went back to Yemen with his wife and six children.”

    “For these five years, none of his family knew anything about him. Everyone believed he was dead. For five years, his family didn’t even know, is he alive or is he dead?”…

    “Muhammad’s mother, Umayma ‘Azzam, separated from her two sons for several years, could not give up hope that Muhammad was still alive. But her brother counseled her to put the matter to rest.” [Heba’s other brother Hussain faced similar difficulties, including rendition and detention].

    “The first news that Muhammad might be alive came on February 28, 2004, five years after his forced transfer to Egypt, when the London-based al-Sharq Al-Awsat broke the story that he was still alive and
    being held in the Tora prison complex. The report was accompanied by a recent photograph of Muhammad.”

    ***
    “After the government acknowledged that Muhammad al-Zawahiri was in custody, it allowed members of his family to visit him in detention. .. During these visits his family learned that Muhammad had been tortured. Mahfuz ‘Azzam told Human Rights Watch that Muhammad’s sister, Heba, a doctor by training, noticed that Muhammad had trouble shaking hands. Heba also saw scars on his wrists, and noted that his feet were swollen. She concluded that the marks were a result of being hung from the ceiling by his wrists.”

    The family’s lawyer was Mamdouh Ismail. Mamdouh Ismail also represented the polymerization expert who studied in North Carolina and had the keys to the apartment used to prepare the 7/7 London bombs. His field was protecting drugs from destruction by enzymes while being delivered to the target organ. He was charged a couple years ago with being a conduct between Zawahiri and jihadis in Iraq, Yemen and Egypt. His intermediary was the Egyptian spymaster and Islamic Group member who wrote about Amerithrax and announced that Islambouli had joined Al Qaeda. Islambouli had led the cell with KSM, who came to be in charge of anthrax planning.

    Following the anthrax mailings, rumors circulated in Islamist circles that Muhammad had been executed. The Americans asked the Egyptian government for a sample of his DNA from the dead body to match it with that of a skull found in Tora Bora, which they suspected was Ayman Al-Zawahiri. The head was delivered to President Bush in a box. “The Egyptian government,” Human Watch explains, “kept quiet, neither confirming nor denying any of the rumors about Muhammad’s fate.”

    “Although Muhammad could not speak freely in front of the prison guards who monitored all of his visits with his family, he asked his mother to make a formal request to the Prosecutor General for a forensics exam. He wanted one to be done as soon as possible, before the marks on his body disappeared. His mother presented the formal request to the government on August 4, 2004;
    the family has yet to receive a response from the government [as of the 2005 publication]. There was also no response from the government to Muhammad’s separate request to be examined by a forensics expert.”

    “In April 2004, Mahfiz ‘Azzam managed to win his first and only visit with his nephew [Muhammad]. The visit lasted only a few minutes, and the entire conversation took place in the presence of the SSI
    liaison officer in Tora prison. In those few minutes, Muhammad briefly conveyed to his uncle glimpses of the torture and ill-treatment that he had endured:”

    “He stayed for four years and half in an underground detention facility run by the mukhabarat, where he did not see sunlight, and could not distinguish between day and night. The interrogation and torture went hand in hand. He lost hope in seeing the sun again.”

    What insights does Tarek Hamouda have about Pharmacology Professor Heba Al-Zawahiri’s ordeal and concern for years over the rendition of her brother Muhammad in 1999 — at which time it was publicly announced that her brother intended to use anthrax against US targets in retaliation? (His transcript would show whether he studied with her).

    Dr. Hamouda and his small company took the millions from DARPA. After his product was used to decontaminate (in testing) the Senate Office Buildings, and his hand cream was pitched to postal workers, these questions are fairly asked given that Attorney Al-Zayat, the blind sheik’s lawyer, had expressly announced at the time of Muhammad Al-Zawahiri’s rendition anthrax would be used against US targets in retaliation of such rendering and mistreatment of Egyptian Movement leaders.

    I explained all of this to the CIA’s Zawahiri Task Force in mid-December 2001. And so any claim of having to play catch-up would be disengenuous. If I knew it, Cheney knew it.

    Does Tarek know Attorney Al-Zayat? Cairo Medical alum and former Vanguards of Conquest leader Dr. Agiza? Cairo Medical alum and former Vanguards of Conquest leader Dr. Al-Sharif? Cairo Medical alum and former Vanguards of Conquest leader Ayman Zawahiri? His childhood friend Tarek Hamid, who consults now for intelligence agencies, reports his own experience of being recruited by Ayman Zawahiri one Friday after classes. He says he got queasy and withdrew when talk turned to burying an Egyptian security officer alive near the mosque. Dr. Hamid, without drawing any inferences, tells me that he called Dr. Hamouda before 911 from abroad and asked about patents — and Dr. Hamouda said it was all in the marketing.

    It’s time to start asking relevant questions — and those questions don’t involve whether Dr. Bruce Ivins found the rituals followed by blindfolded co-eds titillating, whether he wrote letters to the editor, or whether he got upset when he was barred from his workplace and his lifelong friends were forbidden to speak to him about what was going on. They don’t involve whether he keyed someone’s car 25 years ago.

    And anyone who does not understand that these questions about Ayman Zawahiri’s anthrax planning need to be respectfully asked and answered — and that FOIA’s obligations are mandatory — is mistaken and is part of the problem, not the solution.

  5. DXer said

    Re microencapsulation

    Note: For a copy of the April 10, 2001 international application, see
    http://www.wipo.int/pctdb/en/wo.jsp?wo=2001072952&IA=US2001040307&DISPLAY=DOCS

    Pub. No.: WO/2001/072952 International Application No.: PCT/US2001/040307
    Publication Date: 04.10.2001 International Filing Date: 16.03.2001
    Chapter 2 Demand Filed: 12.09.2001
    IPC: C12M 3/00 (2006.01)
    Applicants: SHEPHERD, Herman, R. [US/US]; (US).
    RUSSELL, Philip [US/US]; (US).
    BAILEY, Charles, L. [US/US]; (US).
    ALIBEK, Ken [US/US]; (US).
    Inventors: BAILEY, Charles, L.; (US).
    ALIBEK, Ken; (US).
    Agent: WHITHAM, Michael, E.; Whitham, Curtis & Christofferson, P.C. Suite 340 11491 Sunset Hills Road Reston, VA 20190 (US).
    Priority Data:
    60/191,771 24.03.2000 US
    Title: MICRODROPLET CELL CULTURE TECHNIQUE
    Abstract: The present invention comprises a novel culture method and device in which living cells are cultured in a plurality of individual microdroplets that are immobilized and isolated within a matrix of hydrophobic particles. The hydrophobic particles adhere to inoculated microdroplets of media, isolating the microdroplets in an aseptic microenvironment. The plurality of individual microdroplets provide an optimal environment for the concentrated growth of cultured cells contained therein.

    (WO/2001/072952) MICRODROPLET CELL CULTURE TECHNIQUE

    MICRODROPLET CELL CULTURE TECHNIQUE DESCRIPTION BACKGROUND OF THE INVENTION Field or ehe Invention The present : invention generally relates to the cultivation and growth of cells on laboratory, pilot plant, or industrial scales and, more particularly, to the cultivation and growth of cells in a plurality of individual microdrcplets of liquid media which are interspersed within a matrix of hydrophobic microparticles.
    ***
    With the surface method, nutrients are absorbed from contact with the media under the culture, oxygen is provided through contact with the air above the culture, and inhibitory metabolites seep down and away from the culture. Surface cultivation of microorganisms has the advantageous features of providing a plentiful oxygen source from the surrounding air and efficient removal cf inhibitory metabolites through absorption from the surface medium. Also, contamination of surface culture can be relatively confined to a minimal surface area of a growing culture.

    On the negative side, surface cultivation of microorganisms is not amenable to large scale production. The process of filling and inoculating numerous individual plates or dishes with culture and then individually harvesting each plate is extremely labor intensive. Furthermore, the storage of solid surface plates or dishes inoculated with microorganisms requires significant allocations of space in sophisticated incubators.

    With the submerged method, a microorganism is cultured throughout the liquid media, Mutrients are absorbed from contact with the media surrounding the individual microorganisms, oxygen and other gasses are provided by various means aeration that one skilled in the art can readily appreciate, and metabolites seep out and into the media. Usually, the nutrient media is also stirred continually, in order to evenly distribute the microorganisms.

    The submerged cultivation process has. the beneficial advantages of being less labor and space intensive than the surface method and can be used to produce large batches of cells in a relatively small space. The submerged method is thus the method of choice currently employed in most pilot and industrial scale production of cultured microrganisms and cells.

    The submerged cultivation method does, however, require an extensive investment in equipment necessary for the large scale production of cell cultures. In addition, the end products that are the object of large scale submerged cultivation (i. e, the intracellular or extracellular metabolic- products of cell and microbe growth) usually require further purification and concentration either from the liquid media or the cells therein. This additional isolation step is necessary because the concentration of product in the media is limited by the metabolites released into the media and the limited solubility of oxygen and/or other gases in the media.

    ***
    According to the invention, cells are cultivated in a plurality of individual microdroplets of liquid media. These microdroplets are created by aerosolizing licuid media that has been inoculated with the cells of interest and coating the aerosolized droplets with hydrophobic particles of solid material, such as silicon dioxide, for example. The individual microdroplets are stabilized within the hydriophobic solid particles, thereby providing a large number of small cell culture reactors. The coated microdroplets each provide a sterile environment for the individual microdroplets contained within the culture. Furthermore, the individual microdroplets each provide an optimum microenvironment with a reduced effect of potentially inhbitory metabolites and optimal accessability to aeration, resulting in substantial increases in the concentration of cells per liquid volume.

    ***
    In one embodiment of the invention, the inoculated media is converted into microdroplets prior to introduction into the coating vessel. Such a process is enabled by introducing the inoculated media via a spray nozzle that dispenses individual microdroplets into the vessel. It is not essential to the practice of the invention that the microdroplets be created prior to the introduction of the droplets into the coating vessel. Thus, in yet another embodiment, the microdroplets are created after the inoculated liquid media is introduced into the coating vessel. Ferromagnetic particles are sterilized and introduced into a non- magnetic mixing/coating vessel. Electromagnetic inductors are mounted in parallel on either side of the coating vessel. Activation of the electromagnetic inductors causes an electromagnetic field to exist within the vessel. Oscillations of this electromagnetic field are induced by the inductors. The ferromagnetic particles orient along and follow the field lines of the elect romagnetic field and follow the oscillations of the field. The rapid motion of the field and particles vigorously mixes the hydrophobic particles and liquid media, inducing the formation of droplets.

    The size of the microdroplets will vary, with an optimum size for the cultivation of microorganisms, for example, usually being between 0. 5 and 2. 0 mm in diameter. Sizes within this range have been found to result in high concentrations of microorganisms per microdroplet. It should readily be understood by one skilled in the art, however, that the optimal size of microdroplet will vary, depending on such factors as the growth rate of the cultured cell type, the amount of optimal aeration for a given cell type, the most effective cell density for productuion of a given metabolite, and the like.

    The size of individual microdrioplets can be regulated by adjusting such factors as the size of the nozzle or portal delivering the liquid or aerosolized media, the volume of the vessel, the speed at which the various components are added, the power and frequency of electromagnetic induction (in one embodiment of the invention), and the type of hydrophobic particle utilized, for example.

    In one particular embodiment of the invention, the vessel is contained in a refrigerated environment to prevent the rapid random motion of the electromagnetic process from destroying the inoculated microdroplets with excessive heat.

    Once the microdroplets of inoculated media have formed. the hydrophobic particles can then intercalate between and around individual microdroplets, creating a semi-liquid slurry comprising a matrix of interspersed micrcdroplets of inoculated culture and hydrophobic particles. In one embodiment of the invention, the particles are pumped into the coating vessel while the ferromagnetic particles and liquid media are agitated, resulting in the simultaneous agitation and mixing of the hydrophobic particles along with the microdroplets. In another embodiment, the hydrophobic particles are introduced through a second opening positioned such that the particles encounter the aerosolized microdroplets of inoculated media as the droplets enter the vessel.

    The hydrophobic particles can be introduce into the vessel by a variety of methods well known within the art, for example, by forced flow with the assistance of an air pump. Introduction of the coating particles can be through the same opening used for the introduction of the inoculated media or through a second opening. The use of two different openings for the media and coating particle introduction may have the advantage of allowing for -easier process controls.

    In one embodiment of the invention, the hydrophobic particles comprises a powder of silicon dioxide. It can readily be seen by one skilled in the art, however, that the hydrophobic particles can alternatively comprise other hydrophobic ceramic particles (e. g., possibly aluminum oxides and zinc oxides).

    In a particularly preferred embodiment, the silicon dioxide particle sare Aerosil 300, produced by Brenntag N. V. of Belgium. In another preferred embodiment, the silicon dioxide particles are selected from the group comprising the AEROSIL series of powders manufactured by the Degussa-hüls Cor ? oration (i.., AEROSIL R 104, AEROSIL R 106, AEROSIL R 202, AEROSIL R 805, AEROSIL R 812, AEROSIL R812. S, AEROSIL R 972, AEROSIL R 974, and AEROSIL R. 8200). Other silicon dioxide particles are contemplated and within the scope of the invention.

    The choice of silicon dioxide particles will vary depending on the organism to be cultured and the amount of aeration required. In general, silicon dioxide particles that are useful in the practice of the present invention will be hydrophobic and have a surface area between 50 and 3EG meters-per gram of weight.

    It si contemplated within the practice of the invention that the percent composition of coating particles to inoculated medium will vary, depending on, but not limited to, such factors as the cell type, the size of the individual microdroplets, and the desired final density and phase of growth that is the objective of the particular culture. In one embodiment of the invention that the ratio of individual coating particles to cultured inoculum may be within a range of 99 : 1 and 1 : 99. In one preferred embodiment of the invention, the ratio individual coating particles to cell inoculum to will be within a range of 1 : 2 to 1.

    Once the micriodroplets are formed and coated, they are evacuated from the coating vessel through narrow slotted openings at the bottom of the vessel.

    In one particular preferred embodiment, the slotted openings will be between 1. 5-2. 0 mm wide but may vary depending on the size of the microdroplets formed. The microdroplets can be as little as 10 to 20 microns, so long as the initial inoculum is dense enough to ensure each microdroplet contains inoculated medium. The microdroplets can be much larger, with diameters greater than 2. 5 mm, so long as the hydrophobic particles are able to maintain the media in individual droplet form. Accordingly, the slots for removal can also be designed to be the same as whatever size the microdroolets are or slightly larger.

    In most cases, the space between the coated microdroplets provides adequate aeration of the cell culture. It is a particularly useful and beneficial feature of the present invention that the space which present : provides an optimum environment for the concentrated growth of cell cultures. The adequate aeration provided with the present invention allows the growing cultures to make optimal use of the liquid media contained within each microdroplet.

    I***
    In one embodiment of the invention, the cultured cells will be micrcorganisms. Fermentatuion of microorganisms can proceed via a batch process or a continuous fermentation process. In the case of batch fermentation, the microdroplets are collected and grown in a fermentation vessel. In a continuous fermentation process, the coated microdroplets are collected from the slots at the bottom of the coating vessel and are grown in long conduits that constitute a fermenting zone. The particular fermentation method used to culture the microdroplets is not critical to the practice of the present invention.

    As can readily be appreciated by on the art, it will not always be necessary or preferable to separate the hydrophobic particles away from the liquid cell culture following cell growth. For example, since silicon dioxide is frequently utilized in soil treatment, there is no need to remove the silicon dioxide from cell 1 cultures that are grown for the purposes of soil treatment. Furthermore, since the hydrophobic particles limit the potential for the spread of contamination, it may be desirable to maintain cultivated cells within the individual hydrophobic microdroplets for storage purposes.

    It is a particularly beneficial feature of a preferred embodiment of the present :- invention that : the enhanced aeration of cultured cells, combined with the efficient removal of metabolites, allow for microbial cultures to divide to a density that consumes all of the available liquid present in a microdroplet. Thus, in a preferred embodiment of the invention there is no need to (1) concentrate cultures or (2) remove the hydrophobic particles from the microdroplet culture. When all of the liquid media is consumed, the hydrophobic particles disassociate from the cell cultures, allowing the cells to interact directly with the surrounding environment.

    Alternatively, once cell growth is complete, the liquid media can be isolated away from the hydrophobic particles through a simple centrifugation step. As can readily be appreciated by one skilled in the art, th etime and force of centrifugation will vary depending on the organism and hydrophobic particle employed in the process.

    The silicon dioxide particles can be sterilized and re-used in another microdroplet cultivation process.

    Example I-Bioremediation The present invention can be used to generate large quantities of a bacterial species to be used for bioremediation methodologies. Many examples of decontamination through bioremediation exist, including bioremediation with Pseudomonas, Nitrobacter and Baccillus strains. See, for example, U. S. Patent 6, 025, 152 to Hiatt for relevant examples of bacterial bioremediation organisms.

    The portability of the present invention allows the production of large numbers of bacteria at the site of need. In addition, the bacteria can be grown on site and released still encased within the hydrophobic particles. Only upon consumption of the nutrient media will the hydrophobic particles dissociate and the bacteria then integrate into the contaminated site. This process ensures that the bacteria will be present in maximum quantities when introduced into the contamination site.

    In a process that employs many of the aspects involved in bioremediation, the present invention can similarly be used in the process of bio- prospecting. This process involves the cultivation of microorganisms used for leaching many precious and rare metals from different ores. Microrganisms such as Thiobacillus thiooxidans and others are used industrially to oxidize sulfide minerals to promote . he process of metal leaching. The following metals might be leached using this process: gold, silver, cooper, germanium, gallium, selenium, indium and many others. Since this method does not require a complicated equipment and high energy consumption, the installation for cultivation can be built at a site of mining.

    EXAMPLE II-Production of Vaccine Products.

    The present invention is suitable to the large scale production of recombinar. t bacteria or tissue culture cells that have been genetically engineered to produce an antigen or antigens that are effective vaccine products. For a relevant example of bacterial vaccine production, see U. S. Patent 6, 036, 593 to Ryan, et al. Bacterial or mammalian cells may be cultivated in a plurality of microdroplets to high densities, eliminating the need to further concentrate the recombinant products of the cultured cells.

    The present invention can be adapted to the large scale growth of recombinant organisms such as yeast cells that have similarly been modified to produce pharmaceutically active proteins such as insulin and other growth factors, as in U. S. Patent 4, 775, 622 to Hitzeman, et al.

    While the invention has been described in terms of a single preferred embodiment, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims.

    • DXer said

      Infect Immun. 2002 April; 70(4): 2022–2028.
      doi: 10.1128/IAI.70.4.2022-2028.2002.

      Mucosal or Parenteral Administration of Microsphere-Associated Bacillus anthracis Protective Antigen Protects against Anthrax Infection in Mice

      Dstl, Chemical and Biological Sciences, Porton Down, Salisbury, Wiltshire, SP4 0JQ,1 School of Pharmacy, University of London, Bloomsbury, London, WC1N 1AX, United Kingdom2
      *Corresponding author. Mailing address: Dstl, Chemical and Biological Sciences, Porton Down, Salisbury, Wiltshire SP4 0JQ, United Kingdom. Phone: 44-1980 614747. Fax: 44-1980 614307. E-mail: HCFSmith@dstl.gov.uk.
      Received July 30, 2001; Revised October 9, 2001; Accepted November 30, 2001.

      Abstract
      Existing licensed anthrax vaccines are administered parenterally and require multiple doses to induce protective immunity. This requires trained personnel and is not the optimum route for stimulating a mucosal immune response. Microencapsulation of vaccine antigens offers a number of advantages over traditional vaccine formulations, including stability without refrigeration and the potential for utilizing less invasive routes of administration. Recombinant protective antigen (rPA), the dominant antigen for protection against anthrax infection, was encapsulated in poly-l-lactide 100-kDa microspheres. Alternatively, rPA was loosely attached to the surfaces of microspheres by lyophilization. All of the microspheric formulations were administered to A/J mice with a two-dose schedule by either the intramuscular route, the intranasal route, or a combination of these two routes, and immunogenicity and protective efficacy were assessed. An intramuscular priming immunization followed by either an intramuscular or intranasal boost gave optimum anti-rPA immunoglobulin G titers. Despite differences in rPA-specific antibody titers, all immunized mice survived an injected challenge consisting of 103 median lethal doses of Bacillus anthracis STI spores. Immunization with microencapsulated and microsphere-associated formulations of rPA also protected against aerosol challenge with 30 median lethal doses of STI spores. These results show that rPA can be encapsulated and surface bound to polymeric microspheres without impairing its immunogenicity and also that mucosal or parenteral administration of microspheric formulations of rPA efficiently protects mice against both injected and aerosol challenges with B. anthracis spores. Microspheric formulations of rPA could represent the next generation of anthrax vaccines, which could require fewer doses because they are more potent, are less reactogenic than currently available human anthrax vaccines, and could be self-administered without injection.

      ***
      Microencapsulation of rPA.

      rPA was entrapped in polymeric microparticles composed of poly-l-lactide (PLLA) (molecular mass, 100 kDa) by using a double emulsion solvent evaporation process. Briefly, lyophilized rPA was dissolved in 0.5 ml of an aqueous solution of 2.5% (wt/vol) polyvinyl alcohol (13 to 23 kDa; 88% hydrolyzed; Aldrich, Dorset, United Kingdom). This primary aqueous phase was vigorously mixed, using a Silverson homogenizer (Silverson Machines, Bucks, United Kingdom), with an oily phase consisting of 0.25 g of PLLA (Polysciences, Warrington, Pa.) dissolved in 5 ml of methylene chloride (CH2Cl2). The resultant water-in-oil emulsion was then added, during vigorous agitation, to 75 ml of 5% (wt/vol) polyvinyl alcohol (13 to 23 kDa; 88% hydrolyzed). Following solvent evaporation overnight, hardened polymeric microparticles were harvested by centrifugation prior to lyophilization.

      DISCUSSION

      For optimum protection against pulmonary anthrax infection it is probably necessary for mucosal pathways, as well as systemic immune pathways, to be primed. One way to achieve good mucosal immunity in the lungs and respiratory tract is to administer the antigen locally, in a particulate form. The presence of rPA as a particulate is critical for enhancing protective immunity when it is administered i.n. In contrast, this study showed that when delivered directly to the respiratory tract epithelium of the mouse, rPA in aqueous solution is nonimmunogenic unless it is combined with a mucosal adjuvant, such as CTB.

      Immunization studies with rPA incorporated into microspheres and gel formers have been described previously (M. Kende, C. Yan, J. Corbett, G. C. Atkins, and S. W. Shalaby, Proc. Int. Symp. Controlled Rel. Bioactivated Mater. 2000, abstr. 0223), and success with microencapsulation has previously been shown in mice with other antigens that protect, for example, against Yersinia pestis infection (3). In this study it was found that for two strains of mice, rPA can be encapsulated in polymeric microparticles without impairment of the immunogenicity and protective efficacy of the protein. In the preliminary trial of the microencapsulated rPA formulation, the circulating anti-rPA IgG concentrations in the formulation 5-immunized mice were approximately one-half those obtained for the mice immunized with free rPA (Table 2). These titers were greater than those in the human anthrax vaccine-immunized mice, which received only one-fifth of a human dose (approximately 0.5 μg of PA, assuming an average vaccine PA content of 2.5 μg per 0.5 ml [13]), 20 times less than the dose of microencapsulated rPA delivered in formulation 5. These data suggest that following i.m. injection, microencapsulated rPA is not processed and presented to the relevant elements in the immune system as effectively as free antigen. It is also feasible that exposure to sheer forces and/or organic solvents during the manufacture of this formulation may have damaged the critical antigenic epitopes of the rPA, reducing its immunogenicity. A number of studies have shown that there is no direct correlation between antibody titer to PA and protection against anthrax infection (12, 22, 25). Despite this, a certain threshold titer of antibody is most likely required for protection, and although microencapsulated rPA formulation 5 was not as immunogenic as free rPA when it was administered by the i.m. route, it induced the threshold titer necessary for protection and produced a level of protection comparable to that provided by the human anthrax vaccine.

      Encapsulation and weak binding of antigen to biodegradable polymers can act as a continuous antigen release system, potentially reducing the number of immunizing doses required to elicit a protective immune response.
      REFERENCES

      4. Fellows, P. F., M. K. Linscott, B. E. Ivins, M. L. M. Pitt, C. A. Rossi, P. H. Gibbs, and A. M. Friedlander. 2001. Efficacy of a human anthrax vaccine in guinea pigs, rabbits and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19:3241-3247. [PubMed]
      5. Friedlander, A. M., P. R. Pittman, and G. W. Parker. 1999. Anthrax vaccine: evidence for safety and efficacy against inhalational anthrax. JAMA 282:2104-2106. [PubMed]
      6. Friedlander, A. M. 1986. Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J. Biol. Chem. 261:7123-7126. [PubMed]

      12. Ivins, B. E., M. L. M. Pitt, P. F. Fellows, J. W. Farchaus, G. E. Benner, D. M. Waag, S. F. Little, G. W. Anderson, P. H. Gibbs, and A. M. Friedlander. 1998. Comparative efficacy of experimental anthrax vaccine candidates against inhalation anthrax in rhesus macaques. Vaccine 16:1141-1148. [PubMed]

      • DXer said

        Kathryn Crockett, Ken Alibek’s assistant — just a couple doors down from Dr. Ali Al-Timimi — addressed these issues in her 2006 thesis, “A historical analysis of Bacillus anthracis as a biological weapon and its application to the development of nonproliferation and defense strategies.” She expressed her special thanks to Dr. Ken Alibek and Dr. Bill Patrick. Dr. Patrick consulted with the FBI and so the FBI credits his expertise. “I don’t want to appear arrogant. I don’t think anyone knows more about anthrax powder in this country,” William Patrick told an interviewer. Dr. Alibek’s access to know-how, regarding anthrax weaponization, similarly, seems beyond reasonable dispute. Dr. Crockett successfully defended the thesis before a panel that included USAMRIID head and Ames strain researcher Charles Bailey, Ali Al-Timimi’s other Department colleague. She says that scientists who analyzed the powder through viewing micrographs or actual contact are divided over the quality of the powder. She cites Gary Matsumoto’s “Science” article in summarizing the debate.

        On the issue of encapsulation, she reports that “many experts who examined the powder stated the spores were encapsulated. Encapsulation involves coating bacteria with a polymer which is usually done to protect fragile bacteria from harsh conditions such as extreme heat and pressure that occurs at the time of detonation (if in a bomb), as well as from moisture and ultraviolet light. The process was not originally developed for biological weapons purposes but rather to improve the delivery of various drugs to target organs or systems before they were destroyed by enzymes in the circulatory system” (citing Alibek and Crockett, 2005). “The US and Soviet Union, however, ” she explains, “used this technique in their biological weapons programs for pathogens that were not stable in aerosol form… Since spores have hardy shells that provide the same protection as encapsulation would, there is no need to cover them with a polymer.“ She explains that one “possible explanation is that the spore was in fact encapsulated but not for protective purpose. Encapsulation also reduces the need for milling when producing a dry formulation.” She wrote: “If the perpetrator was knowledgeable of the use of encapsulation for this purpose, then he or she may have employed it because sophisticated equipment was not at his disposal.”

        One military scientist who has made anthrax simulants described the GMU patents as relating to an encapsulation technique which serves to increase the viability of a wide range of pathogens. More broadly, a DIA analyst once commented to me that the internal debate seemed relatively inconsequential given the circumstantial evidence — overlooked by so many people — that US-based supporters of Al Qaeda are responsible for the mailings. Most of Dr. Ivins’ colleagues have thought Al Qaeda was responsible.

        Clarifying the matter — or not — Sandia’s Joseph Michael told FOX News, “I don’t think this exonerates (Ivins) at all.” He added, “I don’t think it’s not enough to say that he did it, as well.”

      • DXer said

        Summary
        Journal of Microencapsulation
        1999, Vol. 16, No. 2, Pages 215-229

        Rhizobacteria microencapsulation: properties of microparticles obtained by spray-drying

        C. Amiet-Charpentier‌

        Rhizobacteria Pseudomonas fluorescens-putida were microencapsulated in Eudragit by spray-drying. These microparticles are subsequently included in seed coating or pelleting material. The survival of the bacterial cell in microparticles were studied under different levels of relative humidity (RH): 0, 33, 55 and 100%. The protective effects of silica, present in certain formulations, were demonstrated at the relative humidities of 33 and 55%. The release of the encapsulated bacteriawas alsostudied over time. The release was fast, the bacteria being observed at 15min immersion of the Eudragit microparticles in an aqueous-buffer medium at 20oC. This result, related tothe physicochemical character of the coating polymer, showed that water was the triggering element for the release of rhizobacteria. Compatibility studies between two film-forming agents used for seed coatings and the encapsulated bacteria, as well as wettability measures of tableted microparticles, were carried out. The bacterial survival was good with the seed coating agent, Sepiret 1039G, and the wettability measurements of agglomerated microparticles were in accord with the rapid release of the microencapsulated bacteria. The application of microparticles containing rhizobacteria on seeds can now be considered for preliminary trials.

      • DXer said

        Bruce wrote in a March 8, 2001 email about a 45-minute presentation about microencapsulation. She said “if you want to have _____________present her microencapsulation work during this 45-minute segment, you can do so, or you can summarize it yourself if you would rather do that.

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