forgive my "flow of consciousness style" but here is my analysis
1- primers- strands of DNA that serve as a starter/template for cloning and PCR reactions
2- degenerate primers--mixtures of similar, but not identical, primers; usually 2 uses for these
a-when trying to amplify genes from a different organism ( genes are similar but not identical)
b-when primer design is based upon protein sequence ( different codons-triad of nucleotides- can code for
the same amino acid and so you cant tell which exact codon is used so you need a mix of them)
Use of these degenerate primers greatly reduce specificity
B.b. strain 2591 is a Ct isolate which as far back as 1988 was compared to various other strains for immunological studies.But nothing about proteins and toxins--are the strains different?? and does the strain 2591 stand as a model for all the other B.b.?
they found a protein of unknown function (BB0755), cloned the gene that they thought should be the progenitor ( recall that using degenerate primers may yield different codons);and they then made another recombinant protein they renamed Bbtox1 ( with so far no proof it was toxin)
In lab tests Bbtox did exhibit toxic behavior BUT what wasnt shown is that this protein is in fact produced by any Borrelia.
we have a slew of codons in our own DNA ; some may even be identical to codons that code for feathers,and that doesnt mean we can or will ever sprout feathers.
their original capture of genes were from those of pertussis and diptheria organisms yet do those produce this type of toxin?
finding a gene sequence and then cloning it and making it produce a protein product doesnt mean the organism can or will produce that product
then I looked at the patent ( thank you Claudia!!)
and i was even more confused--its basically them patenting the method and the end product of a peptide (protein) that it seems they want to inject to induce an antibody response--ie VACCINE
the patent talks a lot about strain B31 ( not 2591) and a lot about the possible ineffectiveness and harm from steere's vaccine of 2 years previous.
the invention bldly proclaims it to be a novel way to diagnose,treat and even prevent lyme diseaase;
here is a key piece:
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated binding polypeptides and binding agents which bind such polypeptides, including antibodies. The foregoing can be used, inter alia, in the diagnosis or treatment of conditions characterized by the expression of a Borrelia burgdorferi toxin, Bbtox1, and by the expression of a Treponema pallidum toxin, Tptox1, nucleic acid or polypeptide.
the whole thing seems to be based upon the presumption of the existence of a toxin they havent yet shown to actually be produced in vivo ( or even in vitro by spirochetes) and the fact it cross reacts to Syphilis???dont they think thats a problem?
so from my perspective--they have this protein which acts like a toxin and they have the genetic sequence that codes for it; but no one can show IF that protein toxin actually is produced anywhere--lab or in animals--by the spirochetes.and the guess about the sequence was taken from pertussis and diptheria organisms that arent similar in behavior, or in the diseases they cause.
the patent also presumes a use for a vaccine based upon this unproven assumption that the protein/toxin is even being produced as well as antibody tests to see if anyone has this. ( have they even tested Lyme pts with CDC positives and neuro sx suggestive of "toxin" and seen if these new tests theyre patenting work?? did they find the "toxin" in pts blood??
it looks to me like they latched onto a protein and patented every possible thing about it--how to find and make it, how to use it etc without having shown that its present and manufactured in spirochetes or in the illnesses they cause.
I am impressed with the level of them yelling "mine, mine, mine" though...every variation of codon and every variation of the protein "toxin" is patented ( did that patent go thru or still pending??)
The polypeptide may be purified from cells/organisms (e.g., Borrelia burgdorferi, Treponema pallidum) which naturally produce the polypeptide by chromatographic means or immunological
no references to show this to be true
NOTE==-many patents declare things that arent proven--the patent office doesnt have to nor does it insure statements are factual--all they really do is make sure its "novel" and a few other things and bam..you own the idea/process/etc
the fact that not much if any of this is in peer reviewed journals leads me to think its not "proven"
A. Detection and Identification of Toxin-producing B. burgdorferi
The present invention also provides methods for detecting Bbtox1 expression, including methods for quantitative analysis of toxin concentration in samples.
in patenting there doesnt have to be proof that a toxin actually exists--they can simply start with the presumption it doeas exist for which : here is the way to find and identify it
cleverly still they "contemplate" treatment with an antitoxin PLUS antibacterials. so who could say which effected successful tx??
they also discuss producing the antitoxin like they do with tetanus--in horses and other large mammals.
methods of introduction of such an antitoxin are "contemplated" not defined--how can they get ownership on "contemplation"??? the old well, maybe we will do this in tablets, maybe by IV and bam they own all ideas??
look at their definitions:
As used herein, the term "toxin" refers to a single protein or peptide that has deleterious effects in cells or subjects. "Bbtox1 toxin activity," "Tptox1 toxin activity," or "toxin activity" as used herein refer to the ability of a single protein or peptide (e.g., Bbtox1, Tptox1) to induce/cause cell death. In important embodiments it is neuronal cell death. In vitro, such activity can be manifested, for example, as ADP- ribosylation and/or elongation factor-2-ribosylation activity (see Examples section). In vivo, such activity is manifested according to any of the symptoms known in the art for Lyme disease and Syphilis.
they claim that any sx known for Lyme or syphilis can be blamed on the toxin that again hasnt been proven to exist from the spirochetes' own production
and after searhcing for references to this toxin I found these:
Polish article that simply states "Neurotoxin, produced by B. burgdorferi Bbtox1 was identified. Encephalopathy signs in Lyme borreliosis could be result of releasing toxico-metabolic products, ability of spirochetes to pass the blood-brain barrier as well as, effect of lymphocytes migration."
but I cant get into their references
and subject index of the 1998 IDSA conference
"Bbtox1 in Lyme disease, abstract 15"
but I cant find it anywhere--was an article ever accepted for publication???
often abstracts at conferences are by authors that are trying to get published but if theyre not accepted into a journal you never see the papers
I DID find this against the idea of toxins in the Borrelia genome
Sequence analysis of the B. burgdorferi genome did not reveal any obvious virulence factors such as toxins. Instead, components exposed on the surface of the organism that are capable of interacting with targets in the host may play key roles in the disease process.
J. Biol. Chem., Vol. 279, Issue 40, 41706-41714, October 1, 2004
if anyone can get hold of this paper it may help
Infection and Immunity, March 2000, p. 1633-1648, Vol. 68, No. 3
Comparative Genome Analysis of the Pathogenic Spirochetes Borrelia burgdorferi and Treponema pallidum
G. Subramanian,1 Eugene V. Koonin,2,* and L. Aravind2
A comparative analysis of the predicted protein sequences encoded in the complete genomes of Borrelia burgdorferi and Treponema pallidum provides a number of insights into evolutionary trends and adaptive strategies of the two spirochete
cmpleted genomes would show if in fact the codes for the protein toxin are there--but it wouldnbt show if theyre able to be turned on and the proeteins made
I was also looking for anything showing Treponema/syph making toxins and my vertigo is hitting again so..thats all for now