Re: Norman - bacteria

[Guest guest]

On Sun, Jan 29, 2006 at 12:59:15AM -0000, wrote:

>> 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?

Mainly I see it as a possibility. (I've never paid the sort of detailed

attention to PCR that I'd pay to it if I were getting my hands dirty with

it myself, or planning to.) I do vaguely recall a couple of examples,

though: to expose DNA from extracellular mycobacteria, they often use

strong chemicals to dissolve the thick, waxy mycobacterial wall; and to

expose DNA from Chlamydia pneumoniae elementary bodies, they often use

chemicals that break disulfide bonds. Intracellular versions of these

classes of bacteria don't have such strong cell walls to break down.

> 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

In that paper, they use a mechanical destruction method to expose the

ribosomal RNA that they're looking for. That seems like the right kind

of method to use in a generalised search for bacteria; with mechanical

destruction, one doesn't have to worry about the chemistry of the

bacterial wall (which differs from species to species), just its

mechanical strength (which also varies, but presumably has some tolerable

maximum value).

They certainly found lots of bacteria, and found them in arthritis

patients rather than controls; the unimpressive part came when they tried

to identify those bacteria: they found them to be a large variety of

miscellaneous bacterial species, most of them not serious pathogens.

>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.

But to that ten percent one has to add all the samples which were PCR

positive for Chlamydia, Borrelia, or Mycoplasma. The abstract doesn't

indicate whether there were tens of those, or thousands.

>Fourth, theres another one out there by the first

>group, this one on ReA.

>----------

That sounds like this one:

http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed & pubmedid=12716447

a quote from which indicates that the sequences used are not completely

universal:

" (Note that the primers R1 and R2 fail to amplify Chlamydia 16S

rRNA, and so Chlamydia sequences would not be expected to be

found in this analysis; their presence is investigated below when

Chlamydia-specific primers were developed and used.) "

Those two primers are the same ones used in their previous work.

They also seem to have gotten unsatisfied with the technique's

performance even on bacteria whose RNA _is_ amplified by those primers,

and supplemented the universal analysis with bacteria-specific primers

for certain of them, too.

Maybe this universal bacteria detection isn't quite ready for prime time,

although it's certainly attractive.

Incidentally, with arthritis, part of how the inflammation is maintained

is neither bacterial nor auto-immune, but mechanical: inflammation

involves swelling in inappropriate places, which increases pressures

(since the joint is designed to work with everything its normal size,

rather than swollen), which then produces more damage, producing more

inflammation. During certain phases even of a bacterial arthritis, this

mechanical feedback process may be all that is going on in a joint.

> [...]

>

>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.

That's a big question, but I'll bet it has a lot of small answers, not

one big one. Shutting things down to keep them from being damaged is a

technique that shows up in all walks of life -- for instance, in

refineries being shut down as Hurricane Katrina approached. The reasons

for it vary widely, depending on what is getting protected by being shut

down. I doubt that biochemistry is less diverse than other areas in this

respect.

--

Norman Yarvin http://yarchive.net

[Guest guest]

> Maybe this universal bacteria detection isn't quite ready for prime

time,

> although it's certainly attractive.

I actually hadnt read that last paper yet, but now have. They state:

" In our previous study in which 46 sequences from each tissue sample

were analysed [14], there was only approximately 80% correlation

between the bacteria detected when the same PCR product was cloned

and sequenced on separate occasions. This indicates that even with a

large amount of sequencing only a relatively small proportion of the

total bacteria present within the joint are detected. "

I'm not sure how the first sentence justifies the second, unless you

assume the PCR has much more than 80% sensitivity in this situation.

They did establish 100% sensitivity of their PCR for amplifying 5 ng

E. coli spiked into the total RNA extract of 200 uL synovial fluid,

but I am unsure whether that could correspond to a higher bacterial

load than what is needed for pathogenesis.

Its not at all clear to me what bacterial load IS needed for

pathogenesis. qtPCR of tertiary syphilis and the polar tuberculoid

form of leprosy would go a long way toward answering the questions,

but no one has done this. In both, organisms are barely findable by

optical microscopy, and in tertiary syphilis at least, they are also

scarcely findable using fluorescent immuno-optical microscopy. But

what about 200-nm-diameter organisms? They are possible and need to

be accounted for in some other way.

At any rate, it is clear that false negatives can happen in PCR. For

example, the joint in arthritis secondary to urogenital chlamydial

infection was once found PCR-neg for chlamydia by at least one

investigation, but was later found PCR+ by many groups (I'll find the

citations if anyone wants). I take these pan-bacterial PCR studies

for what their worth, which is considerable tho not absolute.

I guess next I will read about the attempts to detect antigens of

killed bacteria in the joint. Unfortunately I may never be able to

assess how good those techniques really are, it being heavy chemistry.

> Incidentally, with arthritis, part of how the inflammation is

maintained

> is neither bacterial nor auto-immune, but mechanical: inflammation

> involves swelling in inappropriate places, which increases pressures

> (since the joint is designed to work with everything its normal

size,

> rather than swollen), which then produces more damage, producing

more

> inflammation. During certain phases even of a bacterial arthritis,

this

> mechanical feedback process may be all that is going on in a joint.

If mechanical damage were the sole driving process at any point,

wouldnt this predict that at that point the disease could be cured by

total immobilization of the joint, combined perhaps with partial

immunosuppression? I dont know if this has been tried.

> That's a big question, but I'll bet it has a lot of small answers,

not

> one big one. Shutting things down to keep them from being damaged

is a

> technique that shows up in all walks of life -- for instance, in

> refineries being shut down as Hurricane Katrina approached. The

reasons

> for it vary widely, depending on what is getting protected by being

shut

> down. I doubt that biochemistry is less diverse than other areas

in this

> respect.

Maybe so. But if static or near-static infections really do cause

alot of disease, I hope theres some kind(s) of broadly conserved

process to disrupt.

[Guest guest]

On Thu, Feb 02, 2006 at 05:53:04PM -0000, wrote:

>> Maybe this universal bacteria detection isn't quite ready for prime time,

>> although it's certainly attractive.

>

>I actually hadnt read that last paper yet, but now have. They state:

>

> " In our previous study in which 46 sequences from each tissue sample

>were analysed [14], there was only approximately 80% correlation

>between the bacteria detected when the same PCR product was cloned

>and sequenced on separate occasions. This indicates that even with a

>large amount of sequencing only a relatively small proportion of the

>total bacteria present within the joint are detected. "

>

>I'm not sure how the first sentence justifies the second, unless you

>assume the PCR has much more than 80% sensitivity in this situation.

Since they don't say exactly what they mean by " 80% correlation " (there

are multiple possible meanings), it'd be unsafe to take that sentence

very seriously.

>> Incidentally, with arthritis, part of how the inflammation is maintained

>> is neither bacterial nor auto-immune, but mechanical: inflammation

>> involves swelling in inappropriate places, which increases pressures

>> (since the joint is designed to work with everything its normal size,

>> rather than swollen), which then produces more damage, producing more

>> inflammation. During certain phases even of a bacterial arthritis,

>> this mechanical feedback process may be all that is going on in a

>> joint.

>

>If mechanical damage were the sole driving process at any point,

>wouldnt this predict that at that point the disease could be cured by

>total immobilization of the joint, combined perhaps with partial

>immunosuppression? I dont know if this has been tried.

Of course. What's been found is that total immobilization is too

extreme, since there are parts of joints which are nourished partly by

the motion of the synovial fluid that occurs when the joint is moved.

But resting a swollen joint is a basic animal response: when it hurts to

use a joint, that's because it is causing damage, so people rest the

joint even without being told to. Rest doesn't necessarily mean avoiding

motion; it may just mean avoiding load-bearing. Nevertheless, one has to

live: the cartilage that is damaged in arthritis takes weeks or months to

heal, not just days, so one can't just stay in bed until it is all

healed; one has to get out of bed and get things done, accepting a

certain amount of damage as a consequence, and/or taking

anti-inflammatories to reduce the swelling and thus the amount of new

damage which is done by using the joint. This is standard medical

therapy. It often produces remission even in people with

bacterial/autoimmune arthritis; it's just that the remission doesn't

last.

--

Norman Yarvin http://yarchive.net