XMRV CFS UK study #II

[kurt]

Professor Malcolm Hooper's opinion of the UK research

We didn't get explicit permission to quote Prof Malcolm Hooper, so I won't quote him directly,
but you can now read his quote on an MEActionUK webpage, here:
http://www.meactionuk.org.uk/xmrv-research-and-the-ramsay-research-fund.htm

(search for the word 'skulduggery', and you'll find his quote)

The actual problem appears to be that the patient characteristics and methodology were not sufficiently outlined in the Science article for outside labs to understand the full issues of finding XMRV. Clearly the points Mikovitz is making now are an attempt to fill in some of those gaps, such as revealing the multiple samples and multiple tests of each sample. She still has not given out the details of the patient sample and that appears essential. And the point that this virus has such a low presence in the WBCs was probably not clear enough.

When you publish in a journal, if you are not revealing everything required for replication, you are obligated to make that fact clear, so outside researchers will know they have to work with you directly to learn the whole story. That was not made clear and a lot of money has been wasted by outside labs that might have been used more productively. This was rare CFS research money that has been wasted! No matter how you spin this, WPI made an error in the way they reported their study, and I hope they correct that soon with an update to their Science article sufficient to allow outside labs to find XMRV on their own (essential to validate the finding). If that does not happen and the negative findings keep piling up WPI and XMRV will lose a lot of credibility.

 

[kurt]

testable is not viable and here you are looking for a very small specific sequence the wpi did not use whole blood they concentrated their protien pre assay from specific cells and further amplified the virus load with culture plus the british blood was taken by phlebotomists with no idea how much care you need to take if you are trying to isolate virus from blood they cant have known they were looking for anything in particular they were ordinary blood samples for routine tests in the uk lysis is a huge problem
the british studies were looking for virus in normal titres

Got it, thanks. However, I have to point out that the blood used by WPI was also from routine sticks (draws), it was banked blood, they happened to have multiple samples available on those patients. They probably were not using live sticks or special precautions. And it is not clear to me exactly how the culturing was different from the PCR, the Science article did not say the PCR test was cultured anyway. The culture study used western blot (antibody). Maybe that was yet another omission.

 

[ukxmrv]

Kurt and all the researchers,

Probably a silly question.

Would it be fair to expect people to go back to the WPI and ask for the full protocol before attempting to replicate the test? Is that maybe, a complete no-no for some reason?

 

[usedtobeperkytina]

Well, I think he is an expert on many retroviruses, not just murine leukemia virus.

But even if he was, just goes to show there is lots of money for all kinds of research. We hear silly study reports all the time.

"Studies show, that a man responds with endorphins in the sexual part of the brain when he sees a woman in a swimsuit." We have all heard of the silly studies like this. why oh why, is it so hard to get money for CFS research? (this was rhetorical).

I don't know enough about studies, etc. But if a flaw was made that not enough details, such as amplification, etc. was not in the Science article, then it seems the editors of Science share some responsibility. That is what they are supposed to do, be a check and balance to what is presented for publication. Also, I don't understand what is needed concerning the cohort. I didn't read the study and I know it was people from Peterson's clinic who are from all over the US, even other countries, who meet the Fakuda or Canadian criteria. The samples were taken at different times and for some, were drawn from four different times. They reflect the gender percentage that is seen in occurrence of CFS. Their average age was higher than what is seen in CFS in general population because these were mostly people that were part of oubreak at Peterson's clinic.

What else needs to be known?

Tina

 

[Hope123]

The problem is how WPI reported the results was not at all standard. While I personally support the WPI's mission and am quite satisfied with the way they have communicated with people with CFS and their advocates, they way they communicated results could be viewed as unprofessional based on my own past experience and comments from my friends in the sciences. I've had to defend the WPI's method of PR to them. With an already controversial illness like this, you want to appear as aboveboard as possible.

I can understand the article in Science being truncated due to its purpose of trying to establish a new human pathogen but

1) if you're going to calculate an odds ratio looking at risk of CFS in people with and without XMRV, the populations, both sick and healthy need to be better described. If not in Science, then an accompanying article. Peterson first described the cohort at the CFSAC meeting which is fine but a political meeting should not be the FIRST venue to talk about this.

2) don't talk about data that isn't published yet or not going to be published anytime soon. The "95%" positive antibody figure was thrown around and is still not published anywhere

3) be very clear about your cohort - the way the data was presented at CFSAC, it sounded like the people with lymphoma were part of the Science study; later WPI site said they were not; also, initially it sounded like the sick cohort was very disabled, then later it was reported they were a wide spectrum. It's OK to correct yourself but do it too often or not clearly and people begin to doubt what you say.

4) I don't know if the WPI or CAA's idea of releasing press releases to each study that comes out is a good idea. I hope these are not put out to the general media. I understand people want to know what is going on but it makes it look like our advocates are heading up a public relations campaign rather than a scientifc search for the truth. It is good for scientists in both groups to write letters to the journals publishing the negative studies -- this is the usual way to debate a study. (I am very happy the NY Times has decided to wait this out and not published the negative study results despite there being journalists there tracking CFS.)

5) Don't release info by bits and pieces here and there. It makes it look like you are intentionally hiding something or tailoring what you say/ do to the moment at hand.

 

[usedtobeperkytina]

From what I have learned, I would agree that the 95% figure would not normally be put out unless in a peer reviewed journal. At the same time, this is a major health concern that involves the blood supply. Just as America was put on alert for H1N1 with little "science" to back up the threat concern, if what the initial evidence indicated was true, then getting the word out was the prudent thing to do. In the case of the CFSAC meeting, I can see that since you showed the association in a peer reviewed publication, additional evidence of stronger association, when this is a major health issue, might need to come out sooner. But I agree, not the standard.

I was listening to a radio program today that was actually encouraging scientists to have more interaction with the news media. I only got bits and pieces of the show, but here it is if you want to listen. It also talks about errors in studies and the process of the search for answers. http://www.sciencefriday.com/program/archives/201002191 The recording is on the top left of the page.

I have no problems with the WPI PR campaign. But they do need to be guarded. And I certainly understand WPI answering news reporter questions when there is a study seemingly conflicting their findings. In fact, I would expect they would answer questions about it. Putting out a press release is no doubt, in response to the public's request for explanation. I think the press releases are fine. Now, I do think it is wrong to attribute malicious motives to other researchers. And Mikovitz did do that concerning the first UK study. She stepped over the edge.

Tina

 

[Cort]

That Japanese study is looking more and more interesting. They are the only other ones I believe that have been able to find XMRV in the blood; what techniques did they use? Did they have to jump through all the hoops the WPI researchers did? Did they have to culture their white blood cells? Look four times using different time points in the unstimulated cells? Maybe Gerwyn or Kurt would know - is it likely they would do that???

How did they find XMRV when the two CFS studies in the UK were not able to?

 

[Cort]

This is from the original science paper:

PBMC were activated by 1 μg/mL PHA (AbbottDiagnostics, Abbott Park, IL) and after 72 hours the cells were cultured with 20
units/mL of IL-2 (Zeptometrix, Buffalo, NY) and subcultured every 3-5 days.

Is this an unusual or usual step in PCR?

This is what they did with the T-cells

After isolation, the CD3+, CD4+ T cells (>95% pure) were cultured in RPMI-1640
medium supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 1 mM
sodium pyruvate and antibiotics. CD4+ T cells were activated by culturing with 20
units/mL of IL-2 and 1 μg/mL PHA.

Cell culture and reagents. Raji, SupT1 and LNCaP were obtained from
American Type Culture Collection (ATCC, Manassas, VA). The cells were
maintained in RPMI-1640 supplemented with L-glutamine (2 mM), penicillin (100
U/mL), streptomycin (100 ng/mL), and FCS (10%) and subcultured 1:5 every 4-5
days.

Do these statements indicate that you have to culture the wbc's first in a special way?

 

[Francelle]

Questions for Dr Mikovits and Dr Cheney!

Earlier in this thread some people were mentioning about asking some questions of Judy Mikovits.

I don't know if anyone has since posted about this but it seems that questions can be sent to the Dr Cheney website for tomorrows Q&A session. The link (and hopefully I am allowed to post it here) is:
http://www.cheneyresearch.com/live?...445-web_broadcast_xmrv_qa_cr&utm_medium=email

 

[julius]

Calf serum?

I read or heard somewhere (think Dr.Mikovits) that XMRV can infect other animals (not mice). But no animals were named specifically. Is there any way there was XMRV in the calf serum supplement?

 

[Cort]

Thanks Francelle,

There is no mention of any culturing in the Groom Study. If culturing is specifically designed to increase the numbers of a very low virus then why did Groom think they could forget this step and still have it work? Did they think their methods were so sensitive that it didn't matter?

 

[Countrygirl]

Calf serum?

I read or heard somewhere (think Dr.Mikovits) that XMRV can infect other animals (not mice). But no animals were named specifically. Is there any way there was XMRV in the calf serum supplement?

One recent study (...thinks....tries to rally memory celll :Sign Please:...nobody home :In bed:....try again later.......it is mentioned on this site ...will need to search) mentioned that cows, dogs, pigs, horses, certain rodents (not mice) can be infected with XMRV. Was it the French...? Calf serum supplement....cows????.....not exactly a million miles apart are they

 

[Angela Kennedy]

The actual problem appears to be that the patient characteristics and methodology were not sufficiently outlined in the Science article for outside labs to understand the full issues of finding XMRV. Clearly the points Mikovitz is making now are an attempt to fill in some of those gaps, such as revealing the multiple samples and multiple tests of each sample. She still has not given out the details of the patient sample and that appears essential. And the point that this virus has such a low presence in the WBCs was probably not clear enough.

When you publish in a journal, if you are not revealing everything required for replication, you are obligated to make that fact clear, so outside researchers will know they have to work with you directly to learn the whole story. That was not made clear and a lot of money has been wasted by outside labs that might have been used more productively. This was rare CFS research money that has been wasted! No matter how you spin this, WPI made an error in the way they reported their study, and I hope they correct that soon with an update to their Science article sufficient to allow outside labs to find XMRV on their own (essential to validate the finding). If that does not happen and the negative findings keep piling up WPI and XMRV will lose a lot of credibility.

Well, Lombardi et al did give quite detailed information about their patient cohort in their supporting Science hosted online material (the link is not working now for some reason, though it was certainly before). In addition to fulfilling BOTH fukuda and Canadian criteria:

"diagnosis of CFS is based upon prolonged disabling fatigue and the presence of cognitive deficits and reproducible immunological abnormalities. These included but were not limited to peturbations of the 2-5A synthetase/RNase L antiviral pathway, low natural killer cell cytotoxicity (as measured by standard diagnostic assyas) and elevated cytokines particularly interleukin-6 and interleukin-8. In addition to these immunological abnormalities, the patients characteristically demonstrated impaired exercise performance with extremely low VO2 max measured on stress testing..." (www.sciencemag.org/cgi/co...)

Scientists planning a 'replication' would have every opportunity to know what sort of patients were being studied, and, they could have asked Mikovits et al if further clarification was needed.

Furthermore, any scientist claiming to specialise in 'CFS' must surely know about the discrepancy between patient cohorts problem. Many 'CFS' sufferers are aware of it! At the very least, both Erlwein et al and Kerr et al should have mentioned the discrepancy. The Canadian Guidelines have been available since 2003, they have been subject to some validation (Jason et al, detailed in my Plosone response), and have been subject to the most studious ignoring I've ever seen in most subsequent research papers on 'CFS'. I think the discrepancies of these two British papers, because Lombardi et al DID use canadian criteria identified cohorts (thereby showing they ARE useful as research criteria) have thrown that problem into stark relief.

I wrote a response to the Erlwein paper, detailing the issue of patient cohorts, here:

http://www.plosone.org/annotation/l...notation/13ea20d1-91e6-49c3-bc4b-8fd1ca18f150

 

[Gerwyn]

Got it, thanks. However, I have to point out that the blood used by WPI was also from routine sticks (draws), it was banked blood, they happened to have multiple samples available on those patients. They probably were not using live sticks or special precautions. And it is not clear to me exactly how the culturing was different from the PCR, the Science article did not say the PCR test was cultured anyway. The culture study used western blot (antibody). Maybe that was yet another omission.

It was draw for virological purposes and thus treated far differently different tubes etc all have an effect on virus load.The techniques used by the WPI are pretty standard for the detection of a viruses in the latent phase seperation of particular cells from whole blood concentrating your suspected stock and so on.The british techniques were not designed for recovering alatent virus with a very low free virus titre -I repeat they were using techniques developed for detecting the aids virus and assumed they could detect any rerovirus that way Without many different amplification protocols you cant isolate a virus that hides only in particular cells.they first isolated these cells and ended up with a concentrated sample to work with The british study didn,t even do this basic step which is a prerequisite for isolating latent viruses.How do you feel about commenting on the british methodology

 

[julius]

Thanks for joining us Angela. I hope you plan on sticking around...we need all the knowledgeable people we can get.

 

[Gerwyn]

The following contains the relevant treatment of blood isolation seperation preservation etc for aids research and so on amplification proceedures The WPi followed the vast majority of these steps and they were not followed in britain



The Laboratory for Viral Immune Pathogenesis of the Department of Experimental Immunology at the AMC studies the pathogenesis of viral infections, in particular HIV infections. We perform both fundamental and patient-related research. Within our diagnostic division we apply and develop laboratory tests for the diagnosis and follow up of acquired immunodeficiency disorders (mainly HIV/AIDS). We also provide high quality laboratory support for pharmaceutical studies and vaccine trials. This includes viable freezing and cryopreservation of peripheral blood mononuclear cells. Over more than 15 years we have acquired vast experience in immunology and virology projects and have an excellent track record for storage and retrieval of cryopreserved samples as part of the Amsterdam Cohort Studies on HIV infection and AIDS and for third parties when additional immunology and/or virology research is required after completion of a trial. We are imbedded in an academic setting allowing direct interaction with highly reputable experts in the fields of virology and immunology.

* Sample processing and cryopreservation
* Immunological tests
* Virological tests
* Host genetic testing
* Sample Logistics
* Collaborations
* Contact

Sample processing and cryopreservation
- Peripheral blood mononuclear cells (PBMC) are isolated from whole blood samples by Ficoll gradient density centrifugation. PBMC are cryopreserved using a Sy-Lab IceCube controlled rate freezer and subsequently stored in liquid nitrogen vapor phase in CryoSolutions MVE 1800 series cryostats.
- Plasma and/or serum is isolated from whole blood by centrifugation and stored at -80C in Sanyo MDF series upright freezers with liquid nitrogen backup.

Immunological tests
- Enumeration of mature human T, B, and natural killer (NK) lymphocytes. Using a Becton Dickinson FACSCanto II we enumerate mature human T (CD3+), B (CD19+), helper/inducer T (CD3+CD4+), suppressor/cytotoxic T (CD3+CD8+), and NK (CD3-CD16+ and/or CD56+) lymphocytes.
>> CD3+ T lymphocytes are a subset of lymphocytes defined by their development in the thymus and by heterodimeric receptors associated with the proteins of the CD3 complex. T cells carry out a variety of functions in an immune response, acting always by interacting with another cell in an antigen-specific manner.
>> CD3+CD4+ helper/inducer T lymphocytes activate macrophages and help B cells produce antibody.
>> CD3+CD8+ suppressor/cytotoxic T lymphocytes kill cells infected with viruses and other intracellular pathogens.
>> CD19+ B lymphocytes are one of the 2 major types of lymphocytes. The antigen receptor on B lymphocytes, usually called the B-cell receptor, is a cell-surface immunoglobulin. On activation by antigen, B cells differentiate into cells producing antibody molecules of the same antigen specificity as this receptor.
>> CD3-CD16+ and/or CD56+ NK cells are large granular, non-T, non-B lymphocytes, which kill virus-infected and some tumor cells. They bear a wide variety of invariant activating and inhibitory receptors, lack antigen-specific receptors. NK cells are important in innate immunity to viruses and other intracellular pathogens, and in antibody-dependent cell-mediated cytotoxicity (ADCC).
- Enumeration of activated human T lymphocytes. CD38 and HLA-DR monoclonal antibodies are used for enumeration of activated (CD38+ and/or HLA-DR+) helper/inducer (CD3+CD4+) and suppressor/cytotoxic (CD3+CD8+) T lymphocytes.
- Enumeration of nave and memory human T lymphocytes. CD45RA and CD27 monoclonal antibodies are used for enumeration of naive memory T lymphocytes. Four subsets can be distinguished based on expression of CD45RA and CD27 antigens:

Subset Phenotype
Nave cells CD45RA+/CD27+
Cytotoxic effector-type cells CD45RA+/CD27-
Memory-type cells CD45RA-/CD27+
Memory/effector-type cells CD45RA-/CD27-

Virological tests
- Determination of HIV-1 co-receptor use using the MT-2 cell line. MT-2 cells are of human origin, more specifically a CD4 positive T-cell lymphoma, which is continuously HTLV-1 infected. The express high levels of the C-X-C chemokine receptor 4 (CXCR4) and hence can be infected by HIV-1 variants that use this receptor alone (X4 variants) or in combination with CCR5 (R5X4 variants). Infection of MT-2 cells results in a cytopathic effect called syncytium formation (fusions between 2 or more cells) that can easily be detected microscopically. HIV-1 variants that infect MT-2 cells are thus scored as syncytium inducing (SI) variants. HIV-1 variants that do not infect MT-2 cells are scored as non-syncytium inducing (NSI) variants. The test is performed by direct co-cultivation of patient-derived peripheral blood mononuclear cells (PBMC) of with MT-2 cells. This culture is maintained for 4 weeks with twice weekly passage and microscopic observation after which the test result is reported.
- Enumeration of the number of productively HIV-1 infected T cells using limiting dilution biological cloning. To determine the number of productively infected cells and to obtain a more complete picture of the diversity of the HIV-1 quasispecies present in an individual in vivo, a virus isolation protocol was developed that allows for the isolation of multiple variants from a single PBMC sample, avoiding overgrowth and loss of slowly replicating variants. The test is performed by limiting dilution co-cultivation of patient-derived PBMC with stimulated healthy donor PBMC in 96-wells plates. This culture is maintained for 4 weeks with weekly passage and testing of viral gag p24 production for each well. After passage, MT-2 cells are used to determine co-receptor usage for each virus producing well. This provides an exact estimate of the number of cells productively infected NSI and SI variants in the sample. Upon request, at the end of the culture individual clones can be expanded and cryopreserved for further characterization.

Host genetic testing
- Determination of C-C chemokine receptor 5 (CCR5) genotype. HIV-1 susceptibility is most obviously determined by the CCR5 genotype and associated ?-chemokine production levels. The CCR5 gene encodes for the co-receptor for R5 HIV-1 variants. People who are homozygous for a 32 bp deletion in CCR5 - which results in a premature stop codon and the absence of functional CCR5 on the cell membrane - are more resistant to HIV-1 infection. HIV-1 infected individuals heterozygous for the 32 bp deletion experience a delayed disease progression compared to individuals carrying only the normal (wild type) alleles. The presence or absence of this 32 bp deletion is rapidly determined by polymerase chain reaction (PCR) amplifying the genomic region encompassing the 32 bp deletion and determining the length of the amplified region using agarose gel electrophoresis.

Sample Logistics
- Sample submission forms. Each sample submitted for processing needs to be accompanied by a completed submission form with unique patient identification information, name and address of the person submitting the sample and the specific services requested. These forms can be provided upon request.
- Sample collection and shipment conditions.
>> Immunological and virological tests require whole blood with the anticoagulant heparin (7-10 mL, green cap tube) and kept at room temperature prior to and during shipment. These samples need to arrive at the lab within 24 hours after blood draw.
>> Genetic testing requires whole blood with the anticoagulant EDTA (7-10 mL, purple cap tube) and kept at room temperature prior to and during shipment. These samples need to arrive at the lab within 48 hours after blood draw.
>> Both anticoagulants can be used for whole blood samples submitted for cryopreservation, but samples must be kept at room temperature and arrive within 24 hours after blood draw.
- Test results. Services can be performed on each work day. Turnaround times depend on the specific service in question, ranging from 1-2 work days for immunological tests to 4 weeks for virological tests. After quality checks and official review, the tests results are provided in writing to the person submitting the sample. If desired, the test result can also be obtained by phone using the telephone numbers provided on the samples submission form.
- Privacy. Our Quality Assurance program includes privacy protection of submitted patient information.

Collaborations
The Laboratory of Viral Immune Pathogenesis has several longstanding national and international collaborations to study host and viral factors in the pathogenesis of HIV-1 infection. The diagnostics lab specifically interacts with:
- International Antiviral Therapy Evaluation Center (IATEC), an international organization for clinical research related to prevention and treatment of HIV and other viral infections
- Municipal Health Service, Amsterdam
- Sanquin Blood Supply Foundation, Amsterdam

Contact
For information about our services please contact:
Agnes Harskamp-Holwerda, 020-5668251, A.M.Harskamp@amc.uva.nl



* Home
* Adres en Route
* Contact
* Vacatures
*

 

[Gerwyn]

blood treatment and virological research methods

The following contains the relevant treatment of blood isolation seperation preservation etc for aids research and so on amplification proceedures The WPi followed the vast majority of these steps and they were not followed in britain



The Laboratory for Viral Immune Pathogenesis of the Department of Experimental Immunology at the AMC studies the pathogenesis of viral infections, in particular HIV infections. We perform both fundamental and patient-related research. Within our diagnostic division we apply and develop laboratory tests for the diagnosis and follow up of acquired immunodeficiency disorders (mainly HIV/AIDS). We also provide high quality laboratory support for pharmaceutical studies and vaccine trials. This includes viable freezing and cryopreservation of peripheral blood mononuclear cells. Over more than 15 years we have acquired vast experience in immunology and virology projects and have an excellent track record for storage and retrieval of cryopreserved samples as part of the Amsterdam Cohort Studies on HIV infection and AIDS and for third parties when additional immunology and/or virology research is required after completion of a trial. We are imbedded in an academic setting allowing direct interaction with highly reputable experts in the fields of virology and immunology.

* Sample processing and cryopreservation
* Immunological tests
* Virological tests
* Host genetic testing
* Sample Logistics
* Collaborations
* Contact

Sample processing and cryopreservation
- Peripheral blood mononuclear cells (PBMC) are isolated from whole blood samples by Ficoll gradient density centrifugation. PBMC are cryopreserved using a Sy-Lab IceCube controlled rate freezer and subsequently stored in liquid nitrogen vapor phase in CryoSolutions MVE 1800 series cryostats.
- Plasma and/or serum is isolated from whole blood by centrifugation and stored at -80C in Sanyo MDF series upright freezers with liquid nitrogen backup.

Immunological tests
- Enumeration of mature human T, B, and natural killer (NK) lymphocytes. Using a Becton Dickinson FACSCanto II we enumerate mature human T (CD3+), B (CD19+), helper/inducer T (CD3+CD4+), suppressor/cytotoxic T (CD3+CD8+), and NK (CD3-CD16+ and/or CD56+) lymphocytes.
>> CD3+ T lymphocytes are a subset of lymphocytes defined by their development in the thymus and by heterodimeric receptors associated with the proteins of the CD3 complex. T cells carry out a variety of functions in an immune response, acting always by interacting with another cell in an antigen-specific manner.
>> CD3+CD4+ helper/inducer T lymphocytes activate macrophages and help B cells produce antibody.
>> CD3+CD8+ suppressor/cytotoxic T lymphocytes kill cells infected with viruses and other intracellular pathogens.
>> CD19+ B lymphocytes are one of the 2 major types of lymphocytes. The antigen receptor on B lymphocytes, usually called the B-cell receptor, is a cell-surface immunoglobulin. On activation by antigen, B cells differentiate into cells producing antibody molecules of the same antigen specificity as this receptor.
>> CD3-CD16+ and/or CD56+ NK cells are large granular, non-T, non-B lymphocytes, which kill virus-infected and some tumor cells. They bear a wide variety of invariant activating and inhibitory receptors, lack antigen-specific receptors. NK cells are important in innate immunity to viruses and other intracellular pathogens, and in antibody-dependent cell-mediated cytotoxicity (ADCC).
- Enumeration of activated human T lymphocytes. CD38 and HLA-DR monoclonal antibodies are used for enumeration of activated (CD38+ and/or HLA-DR+) helper/inducer (CD3+CD4+) and suppressor/cytotoxic (CD3+CD8+) T lymphocytes.
- Enumeration of nave and memory human T lymphocytes. CD45RA and CD27 monoclonal antibodies are used for enumeration of naive memory T lymphocytes. Four subsets can be distinguished based on expression of CD45RA and CD27 antigens:

Subset Phenotype
Nave cells CD45RA+/CD27+
Cytotoxic effector-type cells CD45RA+/CD27-
Memory-type cells CD45RA-/CD27+
Memory/effector-type cells CD45RA-/CD27-

Virological tests
- Determination of HIV-1 co-receptor use using the MT-2 cell line. MT-2 cells are of human origin, more specifically a CD4 positive T-cell lymphoma, which is continuously HTLV-1 infected. The express high levels of the C-X-C chemokine receptor 4 (CXCR4) and hence can be infected by HIV-1 variants that use this receptor alone (X4 variants) or in combination with CCR5 (R5X4 variants). Infection of MT-2 cells results in a cytopathic effect called syncytium formation (fusions between 2 or more cells) that can easily be detected microscopically. HIV-1 variants that infect MT-2 cells are thus scored as syncytium inducing (SI) variants. HIV-1 variants that do not infect MT-2 cells are scored as non-syncytium inducing (NSI) variants. The test is performed by direct co-cultivation of patient-derived peripheral blood mononuclear cells (PBMC) of with MT-2 cells. This culture is maintained for 4 weeks with twice weekly passage and microscopic observation after which the test result is reported.
- Enumeration of the number of productively HIV-1 infected T cells using limiting dilution biological cloning. To determine the number of productively infected cells and to obtain a more complete picture of the diversity of the HIV-1 quasispecies present in an individual in vivo, a virus isolation protocol was developed that allows for the isolation of multiple variants from a single PBMC sample, avoiding overgrowth and loss of slowly replicating variants. The test is performed by limiting dilution co-cultivation of patient-derived PBMC with stimulated healthy donor PBMC in 96-wells plates. This culture is maintained for 4 weeks with weekly passage and testing of viral gag p24 production for each well. After passage, MT-2 cells are used to determine co-receptor usage for each virus producing well. This provides an exact estimate of the number of cells productively infected NSI and SI variants in the sample. Upon request, at the end of the culture individual clones can be expanded and cryopreserved for further characterization.

Host genetic testing
- Determination of C-C chemokine receptor 5 (CCR5) genotype. HIV-1 susceptibility is most obviously determined by the CCR5 genotype and associated ?-chemokine production levels. The CCR5 gene encodes for the co-receptor for R5 HIV-1 variants. People who are homozygous for a 32 bp deletion in CCR5 - which results in a premature stop codon and the absence of functional CCR5 on the cell membrane - are more resistant to HIV-1 infection. HIV-1 infected individuals heterozygous for the 32 bp deletion experience a delayed disease progression compared to individuals carrying only the normal (wild type) alleles. The presence or absence of this 32 bp deletion is rapidly determined by polymerase chain reaction (PCR) amplifying the genomic region encompassing the 32 bp deletion and determining the length of the amplified region using agarose gel electrophoresis.

Sample Logistics
- Sample submission forms. Each sample submitted for processing needs to be accompanied by a completed submission form with unique patient identification information, name and address of the person submitting the sample and the specific services requested. These forms can be provided upon request.
- Sample collection and shipment conditions.
>> Immunological and virological tests require whole blood with the anticoagulant heparin (7-10 mL, green cap tube) and kept at room temperature prior to and during shipment. These samples need to arrive at the lab within 24 hours after blood draw.
>> Genetic testing requires whole blood with the anticoagulant EDTA (7-10 mL, purple cap tube) and kept at room temperature prior to and during shipment. These samples need to arrive at the lab within 48 hours after blood draw.
>> Both anticoagulants can be used for whole blood samples submitted for cryopreservation, but samples must be kept at room temperature and arrive within 24 hours after blood draw.
- Test results. Services can be performed on each work day. Turnaround times depend on the specific service in question, ranging from 1-2 work days for immunological tests to 4 weeks for virological tests. After quality checks and official review, the tests results are provided in writing to the person submitting the sample. If desired, the test result can also be obtained by phone using the telephone numbers provided on the samples submission form.
- Privacy. Our Quality Assurance program includes privacy protection of submitted patient information.

Collaborations
The Laboratory of Viral Immune Pathogenesis has several longstanding national and international collaborations to study host and viral factors in the pathogenesis of HIV-1 infection. The diagnostics lab specifically interacts with:
- International Antiviral Therapy Evaluation Center (IATEC), an international organization for clinical research related to prevention and treatment of HIV and other viral infections
- Municipal Health Service, Amsterdam
- Sanquin Blood Supply Foundation, Amsterdam

Contact
For information about our services please contact:
Agnes Harskamp-Holwerda, 020-5668251, A.M.Harskamp@amc.uva.nl



* Home
* Adres en Route
* Contact
* Vacatures
*

 

[Angela Kennedy]

Thanks for joining us Angela. I hope you plan on sticking around...we need all the knowledgeable people we can get.

Hi Julius - that's very kind of you! : )

I think there are a LOT of knowledgeable people in the community. There is a dissonance between what many in the community understand, and the apparent lack of this by some of the 'scientists'. This is not meant to be inflammatory, but I do think we need to stop excusing scientists for not establishing more 'Canadian' cohorts in ME/CFS research, or at least acknowledging, in the sections of their papers detailing 'limitations of study', the discrepancies between those cohorts like the 'empirical' CDC and Wessley/White Oxford/minimal Fukuda criteria cohorts, and people who should be included in a Canadian defined research cohort, perhaps (I'd say definitely) with the additional abnormal physiological responses detailed in the Lombardi paper. I've questioned the Kerr team about this in my (still not published) response to the retrovirology paper on biomed central.

The Lombardi project, whatever happens with XMRV, has given the world of ME/CFS research a Canadian defined cohort, shown it is possible to use Canadian criteria usefully (in addition to the validation paper by Jason et al), and therefore in this respect, I consider the Lombardi project a major scientific 'breakthrough', apart from the XMRV findings which obviously have been rightfully focused upon.

 

[Gerwyn]

Hi Julius - that's very kind of you! : )

I think there are a LOT of knowledgeable people in the community. There is a dissonance between what many in the community understand, and the apparent lack of this by some of the 'scientists'. This is not meant to be inflammatory, but I do think we need to stop excusing scientists for not establishing more 'Canadian' cohorts in ME/CFS research, or at least acknowledging, in the sections of their papers detailing 'limitations of study', the discrepancies between those cohorts like the 'empirical' CDC and Wessley/White Oxford/minimal Fukuda criteria cohorts, and people who should be included in a Canadian defined research cohort, perhaps (I'd say definitely) with the additional abnormal physiological responses detailed in the Lombardi paper. I've questioned the Kerr team about this in my (still not published) response to the retrovirology paper on biomed central.

The Lombardi project, whatever happens with XMRV, has given the world of ME/CFS research a Canadian defined cohort, shown it is possible to use Canadian criteria usefully (in addition to the validation paper by Jason et al), and therefore in this respect, I consider the Lombardi project a major scientific 'breakthrough', apart from the XMRV findings which obviously have been rightfully focused upon.

I agree wholeheartedly with both posts

 

[Bob]

Well, Lombardi et al did give quite detailed information about their patient cohort in their supporting Science hosted online material (the link is not working now for some reason, though it was certainly before). In addition to fulfilling BOTH fukuda and Canadian criteria:

...I wrote a response to the Erlwein paper, detailing the issue of patient cohorts, here:
http://www.plosone.org/annotation/l...notation/13ea20d1-91e6-49c3-bc4b-8fd1ca18f150

...The Lombardi project, whatever happens with XMRV, has given the world of ME/CFS research a Canadian defined cohort, shown it is possible to use Canadian criteria usefully (in addition to the validation paper by Jason et al), and therefore in this respect, I consider the Lombardi project a major scientific 'breakthrough', apart from the XMRV findings which obviously have been rightfully focused upon.

Hi Angela,
A big welcome to the forum. As you might have seen, one of your responses to the UK studies has already been commented on, earlier in this discussion thread.
I'm glad that you've joined the forum! it's great to have you add your insights here.
And it really is a great forum isn't it.

I appreciated your insightful comparison of the patient cohorts.
From what you say, I think I understand that the WPI went out of their way to exclude non-organic illnesses, whereas Wessely & Co went out of their way to exclude organic illnesses. So they were completely opposite patient cohorts.
I hope that research scientists will now learn from the UK studies that diagnostic criteria is crucial.

One thing that I'm not clear on, and I wonder if anyone can clear this up for me?
The WPI study says they selected patients using the Fukuda and Canadian criteria...
does this mean that each patient has to fulfill both criteria,
or does it mean that that some patients fulfilled the Fukuda definition, and some the Canadian definition?
Although, I think that this question is a little redundant, because the WPI were testing for physical signs of organic illness anyway, so they were basically narrowing down the patients to an organic illness which has an effect on the body at the cellular level.
Bob