Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

Can you use flow cytometry for calculating viral load? Just make
antibodies against gp120 and/or gp41, and link 'em to fluorophores.

And could this be a possible treatment for AIDS: synthesize a peptide
nucleic acid --which is much more resistant to degradation, since
neither nucleases nor proteases recognize it-- which is the antisense
strand for the mRNA of the 'env' gene --or the reverse transcriptase
gene, or the integrase gene, or the...--. Then, link the PNAs to cell
penetrating peptides, encapsulate them into liposomes and link the
liposomes to antibodies against CD4. Then patent it, test it on HIV
infected mice, once some promising results are obtained, apply for a
BLA, and start clinical trials. I predict it should be a very safe
drug, since the drug should theoretically only be present in CD4+ T-
cells. It should also be very effective since gp41 isn't just
inhibited, like w/ enfuvirtide, it can't even be produced any more,
since there's no readable instructions. Thus, the HIV viral load
should drop preciptiously, maybe even to 0 RNA copies/ml. It would
have to be given by injection, since the liver would most likely
degrade the drug.

Could this treatment work?

Thanks!!!

Re: Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

On Thu, 8 May 2008 14:16:28 -0700 (PDT), douglas
<Protoman2050@gmail . com > wrote:

>Can you use flow cytometry for calculating viral load? Just make
>antibodies against gp120 and/or gp41, and link 'em to fluorophores.
>


Ah, so this is about HIV.

Measuring free virus?

That is a reasonable question, and it is just a technical point as to
whether or not it will work. The concept sounds fine. The key point
may be whether the labeled virus is a big enough target for the
detector.

What is standard now? Measuring RNA? I suspect that is easier --
requires less fancy equipment.


>And could this be a possible treatment for AIDS: synthesize a peptide
>nucleic acid --which is much more resistant to degradation, since
>neither nucleases nor proteases recognize it-- which is the antisense
>strand for the mRNA of the 'env' gene --or the reverse transcriptase
>gene, or the integrase gene, or the...--. Then, link the PNAs to cell
>penetrating peptides, encapsulate them into liposomes and link the
>liposomes to antibodies against CD4. Then patent it, test it on HIV
>infected mice, once some promising results are obtained, apply for a
>BLA, and start clinical trials. I predict it should be a very safe
>drug, since the drug should theoretically only be present in CD4+ T-
>cells. It should also be very effective since gp41 isn't just
>inhibited, like w/ enfuvirtide, it can't even be produced any more,
>since there's no readable instructions. Thus, the HIV viral load
>should drop preciptiously, maybe even to 0 RNA copies/ml. It would
>have to be given by injection, since the liver would most likely
>degrade the drug.


If I understand this, it is basically a type of antisense treatment,
to prevent HIV production from an infected cell. yes?

One barrier to any such treatment is the "reservoir" problem --
virus-infected cells that just hide during treatment, and then come
out later.

Beyond that... Sounds logical. I'm not close enough to actual work to
evaluate it and compare it with things that have been done. It is a
fairly complex procedure -- which means a lot of assumptions are being
made about what is happening, and a lot of steps to work out. Often
the only way to know is to try it.

Have you looked in PubMed for things of this type?

bob

Re: Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

On May 8, 9:02 pm, Bob <bbx107....@excite.XYZ . com > wrote:
> On Thu, 8 May 2008 14:16:28 -0700 (PDT), douglas
>
> <Protoman2...@gmail . com > wrote:
> >Can you use flow cytometry for calculating viral load? Just make
> >antibodies against gp120 and/or gp41, and link 'em to fluorophores.
>
> Ah, so this is about HIV.
>
> Measuring free virus?
>
> That is a reasonable question, and it is just a technical point as to
> whether or not it will work. The concept sounds fine. The key point
> may be whether the labeled virus is a big enough target for the
> detector.
>
> What is standard now? Measuring RNA? I suspect that is easier --
> requires less fancy equipment.
>
>
>
>
>
> >And could this be a possible treatment for AIDS: synthesize a peptide
> >nucleic acid --which is much more resistant to degradation, since
> >neither nucleases nor proteases recognize it-- which is the antisense
> >strand for the mRNA of the 'env' gene --or the reverse transcriptase
> >gene, or the integrase gene, or the...--. Then, link the PNAs to cell
> >penetrating peptides, encapsulate them into liposomes and link the
> >liposomes to antibodies against CD4. Then patent it, test it on HIV
> >infected mice, once some promising results are obtained, apply for a
> >BLA, and start clinical trials. I predict it should be a very safe
> >drug, since the drug should theoretically only be present in CD4+ T-
> >cells. It should also be very effective since gp41 isn't just
> >inhibited, like w/ enfuvirtide, it can't even be produced any more,
> >since there's no readable instructions. Thus, the HIV viral load
> >should drop preciptiously, maybe even to 0 RNA copies/ml. It would
> >have to be given by injection, since the liver would most likely
> >degrade the drug.
>
> If I understand this, it is basically a type of antisense treatment,
> to prevent HIV production from an infected cell. yes?
>
> One barrier to any such treatment is the "reservoir" problem --
> virus-infected cells that just hide during treatment, and then come
> out later.
>
> Beyond that... Sounds logical. I'm not close enough to actual work to
> evaluate it and compare it with things that have been done. It is a
> fairly complex procedure -- which means a lot of assumptions are being
> made about what is happening, and a lot of steps to work out. Often
> the only way to know is to try it.
>
> Have you looked in PubMed for things of this type?
>
> bob- Hide quoted text -
>
> - Show quoted text -

Wow...someone who takes me seriously. Thanks, Mr Bob --or is it Dr? Or
Prof?--.

Yes, it is antisense. Either antisense or RNAi, I can't predict what
the pathway the biologic will trigger in vivo. Can you --or someone
else-- fill me in, b/c this is basically still a thought experiment,
as I've just entered community college as a freshman --I'm 16.5--, so
I've got no access to the high powered lab needed to do such an
experiment for real. Maybe for my PhD or MD dissertation... If I'm
really lucky, maybe for my BSc senior project. Considering my age and
academic rank, am I intelligent for my age, or below average? I plan
on being a physician-scientist.

Do you think targeting the reverse transcriptase, or integrase,
instead of or in addition to the env gene would be more effective? And
what about my plan to use peptide nucleic acid, instead of RNA or DNA?
And what of packaging the biologic into liposomes studded w/ anti-CD4
antibodies? Or would using cell penetrating peptides alone be more,
um, cost-effective? I now realize that using both would be a
redundant waste of money.

Do you think this would be used as a last resort drug, when everything
else failed? Or would it be first-line?

And, just for fun, what do you think the generic and brand name of
this biologic would be?

Which would be more sensitive and specific, PCR or flow cytometry for
diagnosing the status of HIV infection? Cheaper?

Thanks!!!!

Re: Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

On Thu, 8 May 2008 23:26:33 -0700 (PDT), douglas
<Protoman2050@gmail . com > wrote:

...

>Yes, it is antisense. Either antisense or RNAi, I can't predict what
>the pathway the biologic will trigger in vivo. Can you --or someone
>else-- fill me in, b/c this is basically still a thought experiment,
>as I've just entered community college as a freshman --I'm 16.5--, so
>I've got no access to the high powered lab needed to do such an
>experiment for real. Maybe for my PhD or MD dissertation... If I'm
>really lucky, maybe for my BSc senior project. Considering my age and
>academic rank, am I intelligent for my age, or below average? I plan
>on being a physician-scientist.
>

Well, that is great. We need both -- and we need scientists who
understand medicine.

You are obviously highly interested and motivated. So, go for it.

(Your scientific ideas are certainly advanced for someone who still
uses a fraction in stating their age. But most should have grown out
of that three years ago. :-) )


It is hard to respond to some of your specifics. There is a real gap
between the thought experiment and reality -- which I think you
realize.

People have been working on HIV for 2-3 decades now. Progress, but no
final answer. A recent vaccine test was quite a failure.

Antisense is so logical, but has never really worked. Delivery is one
problem. Specificity is another.

RNAi is fairly new. Very promising, and some things are in clinical
tests. Yet there are concerns -- of the same general types. One recent
paper showed that what was intended as RNAi was working without even
entering the cell -- simply being recognized by a TLR as double
stranded RNA. Whether this is good news or bad news is not clear, but
it certainly is news.

So, lots of logical ideas. Then we need to try them -- and tune them.
And see if any work in practice.


Suggestion...

Go to PubMed. Search on

HIV antisense

or

HIV RNAi

or of course your own variations.

Each of these will give you many "hits". Check that the output is
sorted by date, so that most recent items are first. One good trick is
to click on the tab for "reviews"; these are articles that summarize
an area, rather than present a single new piece of work. Both are
good, but reviews can be a good way to get started, to get a feel for
the field.

Then, browse as you wish.

For each item listed where the title intrigues you, click on it, and
you will get the abstract. Browsing 10-20 abstracts doesn't take too
long, and can give you some feel for the current status.

If you want an entire article, click on "links". You may or may not
find that you have access; some things are freely available, some are
not.

Remember, as you read articles, they can get highly technical, and can
easily be intimidating. They can also take a lot of time, so you want
to learn to get the highlights. Just reading the abstract, then the
intro and discussion, will give you the main ideas, including where
the work fits into the big picture. Articles are different from
textbooks! Don't just try to read them straight thru. In fact, you may
rarely read the detailed materials and methods (unless you are
actively working in the field), and you may or may not want to read
the results in detail. You use some judgment about how much time to
spend on each article -- vs looking at more articles (not to mention
whatever else you do with your life).


I've posted some info on using PubMed on my page:
* w w w .geocities . com /b_bruner/library.htm
But I suspect what I wrote above is probably enough; just explore.


The idea here is to take all these ideas you have, which show that you
are interested and know much about the system, and now build on
that... read what people are really doing now. And again, caution, it
can get messy.


Do you have access to a university library? If so, you can probably
get more articles by using their library.

I have also sent you a private email -- assuming your address shown is
valid.


Ok, if I try to answer everything, I'll never finish. So I'll post at
this point. I'll try to discuss the cell sorting vs PCR another time.

bob

Re: Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

On Thu, 8 May 2008 23:26:33 -0700 (PDT), douglas
<Protoman2050@gmail . com > wrote:

Ok, I said i would tackle this soemtime.

>
>Which would be more sensitive and specific, PCR or flow cytometry for
>diagnosing the status of HIV infection? Cheaper?


My intuition is that PCR will win -- on all (most?) counts. Let me
elaborate on my thinking, which is more or less an educated guess.
That way, you can pursue individual points if you want to, rather than
just the bottom line.


Cost can be tricky. Flow cytometry still involves a rather expensive
device, I am fairly sure. Now, if you happen to have one, not fully
utilized, then using it doesn't cost much. But if you have to buy it,
it is a big deal.

Cost of supplies is probably considerably lower for PCR. Making
primers is now routine. Producing antibodies is not.


Sensitivity. PCR is in principle ultra-sensitive, down to "one". That
is not something other techniques can reach. Of course, amplification
is why it works on "one". And people do try to incorporate
amplification into other methods.


Note that there may be different answers for different situations. One
situation is routine clinical use. Another is research use. They have
different needs.


Ok, some thoughts, with some reasons.

bob

Re: Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

On May 14, 9:01 pm, Bob <bbx107....@excite.XYZ . com > wrote:
> On Thu, 8 May 2008 23:26:33 -0700 (PDT), douglas
>
> <Protoman2...@gmail . com > wrote:
>
> Ok, I said i would tackle this soemtime.
>
>
>
> >Which would be more sensitive and specific, PCR or flow cytometry for
> >diagnosing the status of HIV infection? Cheaper?
>
> My intuition is that PCR will win -- on all (most?) counts. Let me
> elaborate on my thinking, which is more or less an educated guess.
> That way, you can pursue individual points if you want to, rather than
> just the bottom line.
>
> Cost can be tricky. Flow cytometry still involves a rather expensive
> device, I am fairly sure. Now, if you happen to have one, not fully
> utilized, then using it doesn't cost much. But if you have to buy it,
> it is a big deal.
>
> Cost of supplies is probably considerably lower for PCR. Making
> primers is now routine. Producing antibodies is not.
>
> Sensitivity. PCR is in principle ultra-sensitive, down to "one". That
> is not something other techniques can reach. Of course, amplification
> is why it works on "one". And people do try to incorporate
> amplification into other methods.
>
> Note that there may be different answers for different situations. One
> situation is routine clinical use. Another is research use. They have
> different needs.
>
> Ok, some thoughts, with some reasons.
>
> bob

Yes. So using PCR is better. And, don't they mass produce mAbs? Can't
you buy them from biotechnology companie, the same ones that make
primers and other stuff?

In usual HIV viral load tests, is the PCR techniqued used sensitive
enough to detect one copy? Bet PCR has 100% specificity, since the DNA
is unique for each virus? So 100% sensitivity and specificity. That
right?

Doug

Re: Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

On Wed, 14 May 2008 23:12:22 -0700 (PDT), douglas
<Protoman2050@gmail . com > wrote:

>On May 14, 9:01 pm, Bob <bbx107....@excite.XYZ . com > wrote:
>> On Thu, 8 May 2008 23:26:33 -0700 (PDT), douglas
>>
>> <Protoman2...@gmail . com > wrote:
>>
>> Ok, I said i would tackle this soemtime.
>>
>>
>>
>> >Which would be more sensitive and specific, PCR or flow cytometry for
>> >diagnosing the status of HIV infection? Cheaper?
>>
>> My intuition is that PCR will win -- on all (most?) counts. Let me
>> elaborate on my thinking, which is more or less an educated guess.
>> That way, you can pursue individual points if you want to, rather than
>> just the bottom line.
>>
>> Cost can be tricky. Flow cytometry still involves a rather expensive
>> device, I am fairly sure. Now, if you happen to have one, not fully
>> utilized, then using it doesn't cost much. But if you have to buy it,
>> it is a big deal.
>>
>> Cost of supplies is probably considerably lower for PCR. Making
>> primers is now routine. Producing antibodies is not.
>>
>> Sensitivity. PCR is in principle ultra-sensitive, down to "one". That
>> is not something other techniques can reach. Of course, amplification
>> is why it works on "one". And people do try to incorporate
>> amplification into other methods.
>>
>> Note that there may be different answers for different situations. One
>> situation is routine clinical use. Another is research use. They have
>> different needs.
>>
>> Ok, some thoughts, with some reasons.
>>
>> bob
>
>Yes. So using PCR is better. And, don't they mass produce mAbs?

yes. But you need to check the price -- and need to know how much you
need.


>Can't
>you buy them from biotechnology companie, the same ones that make
>primers and other stuff?
>
>In usual HIV viral load tests, is the PCR techniqued used sensitive
>enough to detect one copy?

I don't have any real work at hand.

Ideally, yes. In practice, it is matter of measuring it. It is
probably known. Search on something like

HIV PCR sensitivity.


>Bet PCR has 100% specificity, since the DNA
>is unique for each virus? So 100% sensitivity and specificity. That
>right?

Has to be checked. Sequences may or may not be unique. They "should"
be, but there always are related sequences around. So need to measure.
(Remember, your genome contains much debris from retroviruses.
Obviously, common primers actually used have been chosen to work
fairly well. The Q may be how much work it took to find them.)

bob

Re: Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

On May 15, 9:00 pm, Bob <bbx107....@excite.XYZ . com > wrote:
> On Wed, 14 May 2008 23:12:22 -0700 (PDT), douglas
>
>
>
>
>
> <Protoman2...@gmail . com > wrote:
> >On May 14, 9:01 pm, Bob <bbx107....@excite.XYZ . com > wrote:
> >> On Thu, 8 May 2008 23:26:33 -0700 (PDT), douglas
>
> >> <Protoman2...@gmail . com > wrote:
>
> >> Ok, I said i would tackle this soemtime.
>
> >> >Which would be more sensitive and specific, PCR or flow cytometry for
> >> >diagnosing the status of HIV infection? Cheaper?
>
> >> My intuition is that PCR will win -- on all (most?) counts. Let me
> >> elaborate on my thinking, which is more or less an educated guess.
> >> That way, you can pursue individual points if you want to, rather than
> >> just the bottom line.
>
> >> Cost can be tricky. Flow cytometry still involves a rather expensive
> >> device, I am fairly sure. Now, if you happen to have one, not fully
> >> utilized, then using it doesn't cost much. But if you have to buy it,
> >> it is a big deal.
>
> >> Cost of supplies is probably considerably lower for PCR. Making
> >> primers is now routine. Producing antibodies is not.
>
> >> Sensitivity. PCR is in principle ultra-sensitive, down to "one". That
> >> is not something other techniques can reach. Of course, amplification
> >> is why it works on "one". And people do try to incorporate
> >> amplification into other methods.
>
> >> Note that there may be different answers for different situations. One
> >> situation is routine clinical use. Another is research use. They have
> >> different needs.
>
> >> Ok, some thoughts, with some reasons.
>
> >> bob
>
> >Yes. So using PCR is better. And, don't they mass produce mAbs?
>
> yes. But you need to check the price -- and need to know how much you
> need.
>
> >Can't
> >you buy them from biotechnology companie, the same ones that make
> >primers and other stuff?
>
> >In usual HIV viral load tests, is the PCR techniqued used sensitive
> >enough to detect one copy?
>
> I don't have any real work at hand.
>
> Ideally, yes. In practice, it is matter of measuring it. It is
> probably known. Search on something like
>
> HIV PCR sensitivity.
>
> >Bet PCR has 100% specificity, since the DNA
> >is unique for each virus? So 100% sensitivity and specificity. That
> >right?
>
> Has to be checked. Sequences may or may not be unique. They "should"
> be, but there always are related sequences around. So need to measure.
> (Remember, your genome contains much debris from retroviruses.
> Obviously, common primers actually used have been chosen to work
> fairly well. The Q may be how much work it took to find them.)
>
> bob- Hide quoted text -
>
> - Show quoted text -

I heard they once used PCR to solve a crime --no, not DNA
evidence...something cooler--; this doctor intentionally infected this
lady w/ HIV and HepC, gotten from his other patient's blood. The
forensic scientists used PCR to determine that the virus they were
infected w/ were identical.

I heard they use it as well to determine whether you were exposed to a
pathogen in a certain area.

Re: Flow cytometry for calculating viral load, and a possibly new treatment for AIDS

On Fri, 16 May 2008 00:16:40 -0700 (PDT), douglas
<Protoman2050@gmail . com > wrote:

>
>I heard they once used PCR to solve a crime --no, not DNA
>evidence...something cooler--; this doctor intentionally infected this
>lady w/ HIV and HepC, gotten from his other patient's blood. The
>forensic scientists used PCR to determine that the virus they were
>infected w/ were identical.
>

Yes.


>I heard they use it as well to determine whether you were exposed to a
>pathogen in a certain area.


Both of these fall into the general class: is the sample like A or
like B? So long as specific base sequence differences are known, then
DNA analysis is suitable. PCR provides sensitivity. Depending on
details, it may provide specificity, or that may come from another
step.

As with any test, one must be aware of possible pitfalls. Simply
showing that the sample appears to be like A does not prove it is A;
perhaps it is C. But if the sample is not like A, then it almost
certainly is not A.


Following 9-11 (2001), there was an anthrax scare. AFAIK, it was never
solved, but part of the analysis was determining which strain of
anthrax was used (hopefully offering some clue as to where it came
from).

People now track flu virus strains, trying to see how they spread.

bob