Norman - bacteria

[Guest guest]

> It still could easily be. One of the problems with PCR is how to

destroy

> the organism, beforehand, so as to expose the DNA. Different forms

of a

> bacterium will naturally have different optimal ways of destroying

them.

Do you know any specifics on this, or do you just see it as a

possibility? I run plasmid extraction alot in my lab (I'm just an

undergrad), which is only on E coli. For that we do a conservative

permeabilization of the E coli envelope, because we want to leave

most of the chromosomes inside the cells and just get the smaller

plasmids out. I havent learned anything about whether it would be

fine to just obliterate the cell envelopes if you wanted to do a

whole-DNA extract.

This is a big concern for me, because with 16S rRNA pan-bacterial

primers, one should be able to look for (at minimum) all bacteria

belonging to every known clade, and maybe all bacteria period (people

seem to think so). Surprisingly, despite the zillions of hours spent

investigating bacteria in idiopathic disease, I cant seem to find any

such pan-bacterial PCR investigations except in arthritides. The

findings arent very impressive and lend one to contemplate abacterial

etiologies, or ones that involve influx of bacterial antigens from

outside the joint. I'd like to have seen these guys do quantitative

PCR to see how many total genomes are in there vs controls. Here are

some notes I made on the findings:

----------

This first group seems pretty judicious (I read full

text), and they investigated RA, OA, and UndiffA:

http://www.pubmedcentral.gov/picrender.fcgi?artid=101566 & blobtype=pdf

Then, second, theres the latter groups ref 61, which

is on UndiffA. I just read the abstract.

Third, theres PMID 11465721 by our own guys, Hudson et

al, up at Wayne State: " DNA in synovial biopsy samples

and SF obtained from 237 patients with various

arthritides, including ReA, rheumatoid arthritis, and

undifferentiated oligoarthritis, was assayed by

polymerase chain reaction (PCR) using " panbacterial "

primers; we chose only samples known to be PCR

negative for Chlamydia, Borrelia, and Mycoplasma

species. [...] Ten percent of patient samples were PCR

positive in panbacterial screening assays. " TEN

percent is not very many.

Fourth, theres another one out there by the first

group, this one on ReA.

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> With PCR, a major problem is contamination. The DNA gets

multiplied so

> vastly that an absolutely miniscule speck of contamination from the

last

> batch can ruin your present one.

True. I like to see microscopy go along with it, which the Balin

alzheimers chlamydia study has (both immuno-optical and IEM).

> > Of course, many with MAC have AIDS, cystic fibrosis,

> >or emphysema, but I'm talking particularly about primary pulmonary

> >MAC - people who are otherwise healthy as far as is known.

>

> They do seem to be pretty old, though; the first paper that popped

up on

> Medline when I typed in " primary pulmonary mycobacterium avium " was

a

> study from Japan, on 72 patients, whose average age was 68. The

immune

> system is much weaker in the elderly.

Yes I saw one paper with an age breakdown. Only a percent or two of

patients were in their 30s.

> Tuberculosis is pretty refractory, too, although not quite that

bad; a

> year or so is the standard treatment length. Even if one is just

> seropositive, with no signs of clinical disease, they tend to treat

for

> at least six months. Sometimes they use one or two drugs, but for

> drug-resistant variants of TB, they use more.

Mitchisons paper " The search for new sterilizing anti-tuberculosis

drugs [not free online] " has tons of info. In active TB, 99%+ of the

Mtb are replicating like hell, and with normal treatment on

nonresistant strains you get a tenfold reduction in bacterial load

every 2 days, early on. The months-long treatment is only needed to

hit the nonreplicating or nearly-nonreplicating cells, or so it is

believed. The latter are far more resistant to bactericidal

chemotherapy, and are probably those within the macrophage

phagosomes. Perhaps in most cases of primary pulmonary MAC (and

Crohns, CFS, MS, etc) the entire bacterial population is like this,

hence the problems.

Why does reproductive stasis protect cells from cidal chemotherapy?

Big question. They seem to also be protected (in Mtbs case) from

things like heat shock death, etc. So its probably something

fundemental. Apparantly there are some chaperone proteins that slow

the rate of damage to functional proteins (whose damage/senesence I

know nothing about) at the cost of slowing their function. Obviously

no bacterium is going to express these constitutively, as a bacterium

that replicates even 1% slower than its fellows is going to be

completely swamped by the latters progeny after 1000 generations. I

dont think a great deal is known yet about such proteins, but at

least they are being studied, particularly by those who want to

eradicate TB (Mtb has no known non-human reservoir). If you could use

this knowledge to make new drugs that waste those non-replicating Mtb

in 2 weeks or so, thatd be it for TB chemotherapy, itd be two weeks

long. It might then become conceivable to start blasting away at

latent TB cases worldwide in a really big way. Mtb work is " charity "

work for the 1st-world research establishment, tho, so it gets a good

amount of dough but not tons and tons. Arthritis on the other hand

has a USA prevelence of like 3%, so alot is spent on it, but without

much attention to bacteria.

However, as far as abx resistance, on the other hand theres also the

Gieffers phenomenon:

http://circ.ahajournals.org/cgi/content/full/103/3/351

I think the Gieffers and/or MAC phenomena probably have alot to do

with whats up in all the semi-abx-treatable diseases. And thats

basically all the ideas/observations Ive soaked up that seem like

they could apply well.