In Vitro vs In Vivo Research / Testing

Topics with information and discussion about published studies related to Lyme disease and other tick-borne diseases.
Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

In Vitro vs In Vivo Research / Testing

Post by Joe Ham » Sun 29 Mar 2009 4:53

Res Microbiol. 1992 Jul-Aug;143(6):629-39.
Interaction of spirochetes with the host.
http://www.ncbi.nlm.nih.gov/pubmed/1475523

Hindersson P, Thomas D, Stamm L, Penn C, Norris S, Joens LA.
Institute of Medical Microbiology, University of Copenhagen, Denmark.

The success of an invading organism must depend on several cytoplasmic, surface-associated and secreted factors. The technical difficulties in handling pathogenic spirochetes like Treponema pallidum and Borrelia burgdorferi have made it difficult to define specific factors involved in entry and long-term survival.

The problem of defining virulence factors has been attacked by several strategies: T. pallidum secretes a number of immunogenic low molecular mass proteins. The most predominant are of molecular weight 15.5 and 22 kDa. Preliminary data suggest that antibodies against these proteins induce protective immunity in rabbits experimentally infected with T. pallidum. Many potentially important surface-associated antigens of T. pallidum have now been cloned and characterized. Two of these, TpD and TpE, are lipoproteins which exhibit characteristic size heterogeneity. The apparent molecular weight of TpE from T. pallidum and T. pertenue are different. The clinical symptoms in syphilis and yaws are very different, but sequence analysis of TpE has shown that the TpE proteins are indeed very similar in the two strains.

This observation makes it unlikely that heterogeneity of TpE can account for the different clinical symptoms of syphilis and yaws. Sequence data for another newly sequenced surface-associated antigen of T. pallidum (molecular weight 41 kDa) indicate that this protein is involved in glucose transport and chemotaxis/motility. Intracellular factors like the molecular chaperonin GroEL have been documented both in treponemes and borreliae. This stress protein is involved in cellular repair processes and folding/assembly of protein subunits. Indirect evidence suggests that GroEL affects the ability of spirochetes to survive in the stressful environment of the infected host.

Several lines of evidence suggest that the Osp proteins of Borrelia are important for host/parasite interaction. Further support for this idea has come from studies of a series of monoclonal antibodies against OspA. A monoclonal antibody against OspA (9B3D) is able to block attachment of B. burgdorferi to a cell monolayer.

Borrelia loses infectivity after several passages in vitro. The loss of pathogenicity is associated with loss of specific plasmids and proteins. One of the low-passage-associated proteins (Lap30) has been cloned and sequenced. Lap30 is a lipoprotein encoded by a 38-kb plasmid, not present in high passage B. burgdorferi.

Aberrant immunological processes induced by the lipopolysaccharide component of Treponema hyodysenteriae could explain the dramatic intestinal lesions in swine dysenteriae. But analysis by TLC reveals that the LPS of this treponeme is different from classical Salmonella LPS.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 1475523 [PubMed - indexed for MEDLINE]

Fin24
Posts: 1699
Joined: Sat 8 Mar 2008 20:14

Re: In Vitro vs In Vivo Research / Testing

Post by Fin24 » Sun 29 Mar 2009 20:36

hmmm
so adding animals to the environment will upscale the passing of borrelia between hosts and vectors so it will also downregulated the pathogenicity eventually--a few to several generations

our zeal to rid us of the deer and rodents is again working against us!!!

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: In Vitro vs In Vivo Research / Testing

Post by Joe Ham » Mon 30 Mar 2009 2:14

Fin wrote:so adding animals to the environment will upscale the passing of borrelia between hosts and vectors so it will also downregulated the pathogenicity eventually--a few to several generations
Excuse me for a minute while I get nit-picky. I stubbed my toe on two words which made awkward reading for me. "Upscale" in my lexicon means hi-rent district, and the past tense of "regulate" didn't seem to fit so I changed them as I reread the passage. If this is a wrong interpretation of what you meant please correct me.

"so adding animals to the environment will increase the passing of borrelia between hosts and vectors so it will also down-regulate the pathogenicity eventually--a few to several generations"

So talking about infectivity only I took exactly the opposite implication from the line:
"Borrelia loses infectivity after several passages in vitro."

I've seen essentially that claim before but can't remember the source. The importance of it is in evaluating research where they are working with a hi-passaged and therefore a weakened strain and then trying to generalize to wild strains that people get directly from ticks.

I took a similar implication from:
"Lap30 is a lipoprotein encoded by a 38-kb plasmid, not present in high passage B. burgdorferi."
Both statements taken together seem to support the notion that wild strains are more virulent (infectious and pathogenic) and therefore in vitro and in vivo are not equivalent. Hence the title of the thread. There are more reasons for that lack of equivalence. Hopefully they will be added so that we can better appraise the credibility of research as it becomes available.
(edit spelling)
Last edited by Joe Ham on Sun 5 Apr 2009 17:50, edited 1 time in total.

Fin24
Posts: 1699
Joined: Sat 8 Mar 2008 20:14

Re: In Vitro vs In Vivo Research / Testing

Post by Fin24 » Mon 30 Mar 2009 20:35

[personal comment removed]
the statement of the researchers was indeed that
"Borrelia loses infectivity after several passages in vitro."

my mind jumped to the HOW can we DO that--how can we get the infectivity to drop--my population biology days--
and to get the infectivity to drop, according to THEM, you need some more passing between host and vector so hence I mused about the fact that we are REMOVING animals and removing the passages, so perhaps we are in fact increasing virulence

and also at the same time, the parallel thought that if we increased the passage by allowing MORE numerous animals interacting--a return of the balanced environment---we may be able to downregulate the virulence

In vivo vs in vitro may have nothing to do with passage, depending upon the methodology--do you know for fact that "most" if not "all" ex vivo methods utilize a higher passage strain and that all en vivo (wild) strains are in fact low passage??in other words separate the parameter of virulence due to passages.

you cant confuse and overlap the 2 different ideas
1. low passage vs high passage when comparing virulence
2.wild vs lab grown comparing both passage and virulence

I believe you can have either low or high passage states in the lab--all you have to DO is take strains that are closest to removed from wilder tick vector ( hasnt yet passed too often between vector and host) for lower passage vs allowing the several rounds of vector-host feeds that some labs use to maintain stock

not every post has to be to exacting standards and its annoying to think you expect others to re-read and proof read especially if they too are functioning at under par!!

and I was so verty-spinny last night I waited until today to even post this and grew weary of thinking I had to keep proofing it

[personal comments removed]
Last edited by Fin24 on Thu 16 Apr 2009 0:29, edited 1 time in total.

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: In Vitro vs In Vivo Research / Testing

Post by Joe Ham » Sun 5 Apr 2009 19:36

Fin wrote:according to THEM, you need some more passing between host and vector so hence I mused about the fact that we are REMOVING animals and removing the passages, so perhaps we are in fact increasing virulence
Our difficulty in talking about this may be due to our apparently different definitions of "passage".
Passage:
The transfer or transplantation of cell, with or without dilution, from one culture vessel to another. It is understood that any time cells are transferred from one vessel to another, a certain portion of the cells may be lost and, therefore, dilution of cells, whether deliberate or not, may occur. This term is synonymous with the term "subculture".
Passage number:
The number of times the cells in the culture have been subcultured or passaged. In descriptions of this process, the ration or dilution of the cells should be stated so that the relative cultural age can be ascertained.
http://www.tissuedissociation.com/glossary.html#passage
I take that to mean "propagation in vitro only". Indeed, passing a bacterium back and forth between the hosts and vectors in which it has become adapted by evolution would seem to be the best way to maintain virulence.

So the difficulty may lie in how "passing" or "passed" is used in common English versus how "passage" or "passaged" is used in the biological sciences. Hindersson et al were careful to emphasize that distinction in the line that you quoted:
"Borrelia loses infectivity after several passages in vitro."

From that you seemed to draw the opposite inference:
to get the infectivity to drop, according to THEM, you need some more passing between host and vector so hence I mused about the fact that we are REMOVING animals and removing the passages, so perhaps we are in fact increasing virulence
I don't see how that follows and therefore I don't see how your next statement is either accurate or relevant.
In vivo vs in vitro may have nothing to do with passage, depending upon the methodology--do you know for fact that "most" if not "all" ex vivo methods utilize a higher passage strain and that all en vivo (wild) strains are in fact low passage?
Isn't "no passage" the definition of wild? - not cultured?

**************************************
ya know not every post has to be to exacting standards and its annoying to think you expect others to re-read and proof read especially if they too are functioning at under par!!
...IMHO if we all try to understand YOU, perhaps you can try to understand "us" without expecting our conformity to your expectations??
Understood, and to some extent agreed because we are all sometimes affected by Lyme fog but some of us seem to take undue advantage of the excuse. How about just the common conventions of English composition that we all learned in grade school such as capitalizing the beginning of sentences and ending with a period. And if proofing your scribblings gets to be too much for you, just don't do it. I will understand. That's why I have a scroll wheel.

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: In Vitro vs In Vivo Research / Testing

Post by Joe Ham » Sun 5 Apr 2009 19:42

Am. J. Trop. Med. Hyg., 43(1), 1990, pp. 87-92

Lyme Borreliosis in Laboratory Animals: Effect of Host Species and in Vitro Passage of Borrelia burgdorferi
http://www.ajtmh.org/cgi/content/abstract/43/1/87

Kathleen D. Moody, Stephen W. Barthold AND Gordon A. Terwilliger
Yale University School of Medicine, New Haven, Connecticut

The susceptibility of several common laboratory animal species to a known pathogenic isolate of Borrelia burgdorferi (N40) was evaluated following intraperitoneal (ip) inoculation of 106–8 spirochetes into 3-day-old Lewis rats, CD-1 mice, Syrian hamsters, and 3-week-old American Dutch rabbits. At 30 days, tissues were cultured for spirochetes and examined histologically.

All species developed multisystemic infection as well as arthritis and carditis, but disease was most severe in rats and mice. In order to evaluate the effect of in vitro passage on the pathogenicity of B. burgdorferi, 3-day-old Lewis rats were inoculated ip with borreliae passaged in culture 2, 5, 11, 17, 21, 26, and 31 times, and evaluated at 30 days by culture, histology, and ELISA antibody titers.

Based upon these parameters, B. burgdorferi (N40) lost its virulence at 17–21 passages.

This study demonstrated that B. burgdorferi was infectious for infant rats, mice, hamsters, and 3-week-old rabbits, although pathogenicity was modulated by host species and the in vitro passage history of the spirochete.

Of the 4 laboratory animal species evaluated in this study, rats and mice appear to have the most potential for further use as animal models of Lyme disease.
What are the implications of this? For example, if a researcher does not specify if he was using a wild isolate or a high passaged lab sourced spirochete would his susceptibility studies be credible?

Fin24
Posts: 1699
Joined: Sat 8 Mar 2008 20:14

Re: In Vitro vs In Vivo Research / Testing

Post by Fin24 » Sun 5 Apr 2009 20:34

How about just the common conventions of English composition that we all learned in grade school such as capitalizing the beginning of sentences and ending with a period
I dont consider chat boards to be under the same convention or conscriptions as an essay required for an academic class--if that were the case far too many would be unable to write here

sorry but we will have to agree to disagree and perhaps be a bit more flexible in your expectations???

as for "passage" have you in fact more closely looked at the methods sections??
sometimes, "in vitro" passage is not limited to culture tube to plate back to tube transfer
to maintain some pathogens, many labs do in fact use vectors and provide " feeds" to/from ticks
I cannot comment on your vs my assumprions until you delve further and confirm methodology for maintaining the pathogens

and yes you can also gauge passage of cultural method denotation vs vectoral method denotation, when comparing up and down regulations of virulence. There are compatible and comparable mechanisms that can and therefore may occur whether youre passing from tube to tube or going from vecotr to host. There are far more degradations and cellular changes when kept entirely in culture, but again its useless to discuss without having the particulars of the source of their cells.and lack of comparisons of what changes when comparing culture method passages vs vecotor-host exchanges

What are the implications of this? For example, if a researcher does not specify if he was using a wild isolate or a high passaged lab sourced spirochete would his susceptibility studies be credible?
more to the point:
1. what are the mechanisms of lost virulence and are they mimicked by in vivo "passage" ( vector to host and back)
2.did they start with "wild" and from where and how was that source maintained? ( within ticks fed off rabbits vs mice, were the ticks originally sterile as in pathogen free, etc)
3. methods of "passage" - subculturing- how done and does that affect outcome
-repeat exposure between feeding ticks and hosts

you can get very caught up in isolating variables and trying to not comnpare apples and oranges but then you get too nitpicky and start worrying about comparing navel oranges with tangerines or worse--mandarin tangerines with clementine tangerines
it becomes hard to discern when and if one needs to be so concerned or if the results can be generalized enough for the acceptance of the conclusions

serial passages in cell cutlure technique-may lead to formation of mutations, degradation of cell communications,etc ( google scholar " negative effects of serial passage techniques of pathogen cell cultures)and so in themselves may be more of a problem than a welcome event

cell cutlure techniques vs vector-host studies using ticks as incubators for the pathogen--has enough been done to elucidate the differences?? I dont think so

here is an intersting and maybe pertinent study
http://aem.asm.org/cgi/content/full/73/19/6045

they trapped rodents and then removed ectoparasites and took the pathogens from within the vecotrs directly to study
Mixed Infections, Cryptic Diversity, and Vector-Borne Pathogens: Evidence from Polygenis Fleas and Bartonella Species
Patrick Abbot,1* Alena E. Aviles,2 Lauren Eller,1 and Lance A. Durden2

wonder what they found in comparison to the pathogens being lab raised?? or if anyone has bothered to see if there are differences

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: In Vitro vs In Vivo Research / Testing

Post by Joe Ham » Thu 9 Apr 2009 17:50

Another mention of in vitro vs in vivo discrepancies.
How many of us are beating ourselves up by using an antibiotic that looks good in vitro but has minimal or no activity in vivo?
However, the in vitro and the in vivo antibiotic susceptibilities of Bartonella do not correlate well for a number of antibiotics; for instance, penicillin has no in vivo efficacy, despite the very low MICs observed in vitro.
http://www.lymeneteurope.org/forum/view ... 502#p12651

cave76
Posts: 3182
Joined: Sun 12 Aug 2007 2:27

Re: In Vitro vs In Vivo Research / Testing

Post by cave76 » Thu 9 Apr 2009 18:50

These results indicate that the in vitro and in vivo activities of cefuroxime against B. burgdorferi are comparable to those of several oral antibiotics currently being used in the treatment of early Lyme disease and suggest that the oral form of this cephalosporin may be an effective alternative therapy for this disease.
Results obtained with four antimicrobial agents in the in vivo hamster model parallel the antibiotic susceptibilities in the in vitro study.
http://www.pubmedcentral.nih.gov/articl ... tid=172012

[personal non contributory, non substantial commentary removed]
Extrapolation from studies with laboratory animals to humans should be done with caution.
http://aac.asm.org/cgi/content/full/46/1/132

But I'll take a remission from an abx that's only good in vitro any ol' day.
Last edited by cave76 on Thu 16 Apr 2009 1:53, edited 1 time in total.

Joe Ham
Posts: 489
Joined: Fri 27 Jul 2007 6:15
Location: New Mexico, USA

Re: In Vitro vs In Vivo Research / Testing

Post by Joe Ham » Thu 9 Apr 2009 21:49

Would it be impudent of me to point out that your first reference quotes of the abstract only are to a 1990 paper that was funded by Glaxo, the manufacturer of cefuroxime (Ceftin)?

Or that the implications that you quoted from the abstract are not supported in the fine print of the full text?:
"the poorest results were obtained with penicillin G, in that no protection was obtained at doses as high as 74.5 mg/kg."

Or that the follow up was extremely short:
"Two weeks after the final antibiotic treatment, the hamsters were sacrificed, and the blood, spleen, kidneys, and bladder were collected and cultured."

And flawed by not even looking at the brain for evidence of a bacterium that is know to be neurotropic?

The full version is available
http://www.pubmedcentral.nih.gov/picren ... obtype=pdf
Cave quoting Wormser wrote:Extrapolation from studies with laboratory animals to humans should be done with caution.
I don't see how that is pertinent to a thread about in vitro vs in vivo research. Maybe in a separate discussion about human vs animal testing which might be very enlightening by exploring different metabolic mechanisms of pro-drugs, different immune responses, etc.
Cave wrote:But I'll take a remission from an abx that's only good in vitro any ol' day.
Maybe that depends on what you mean by "remission". If you mean just feeling better that can be accomplished with just about any antibiotic that is effective against Bb spirochetes peripherally. Even if that abx only succeeds in driving and holding the spirochete into the cyst form such as ceftriaxone.

But what about "remission" in the brain? It has no pain sensors, ya know, and therefore symptoms of brain damage only become apparent by mental and psychological symptoms after the damage is done.

Compare tertiary syphilis symptoms
and see also the thread Lyme and Alzheimer's.
and the thread started by you The Emerging Role Of Infection In Alzheimer's Disease
I worry about you because you wouldn't have made that mistake (oversight) in the past; you started that thread only about a year ago.

Post Reply