Interesting CFS research

[Guest guest]

Interesting....but with the Immune Modulators (if research funded) we won't

need to know exactly what or why the immune system is off.

Cheryl

http://www.ivpresearch.org/cfs0.cfm

Relationships Between Human T-Lymphotropic Virus Type II (HTLV-2) and Human

Lymphotropic Herpesviruses in Chronic Fatigue Syndrome

Project funded by The National CFIDS Foundation, Inc.; Needham,

Massachusetts

Background: In 1991 DeFreitas et al. (1) from the Wistar Institute in

Philadelphia reported the detection of HTLV-2 gag gene related DNA

sequences in the lymphocyte DNA of patients with CFS [72% of pediatric

patients and 83% of adult patients] by means of the newly developed

polymerase chain reaction (PCR) technique. DNA samples from healthy control

individuals were negative for HTLV-2 gag gene related DNA sequences.

Several aspects of these studies deserve comment.

Strengths of this work include:

• use of a " hot start " PCR technique (2) that maximized the efficiency of

the PCR reaction (Appendix 1)

• the study of well characterized and very ill CFS patients who met the

1988 CDC criteria for CFS (3)

• use of an upstream PCR DNA primer whose sequence would allow equivalent

detection of both the A and B types of HTLV-2.

Weaknesses of these studies include:

• the use of a high GC content (85% GC) DNA probe for detection of the PCR

product

• use of a downstream PCR DNA primer sequence that might specifically fail

to detect the B type of HTLV-2.

Since 1991 there have been five additional published reports of studies of

HTLV-2 in CFS. These papers were identified by a search of the National

Library of Medicine for the MESH terms " fatigue syndrome, chronic " and

" retroviridae " . Of these reports, one was a non-peer reviewed survey

published in the Morbidity Mortality Weekly Report of the Centers for

Disease Control and Prevention (CDC) (4). Although it is not stated, it is

implied that one of the laboratories involved in this study was that of

DeFreitas et al. Using the PCR technique with the original DeFreitas et al.

primers, no differences were found between CFS patients and controls with

respect to the detection of HTLV-2 DNA sequences in lymphocyte DNA

(approximately 60% of both groups were positive for HTLV-2 gag gene related

DNA sequences). The main strength of this study was that the samples were

analyzed in blinded fashion.

Weaknesses of this report include:

• failure to use a " hot start " PCR procedure such as that used by DeFreitas

et al.

• the laboratories involved in the study are not identified

• no technical details concerning the procedures used are provided

(especially with respect to the " modification " of the PCR assay)

• the patients are not clinically described except that they met the 1988

CDC criteria for CFS.

Another of these five reports is that of Gow el al from laboratories in

Scotland and Germany (5). Again using the original DeFreitas et al.

primers, as well as another HTLV-2 primer pair, no differences were

observed between CFS patients and control subjects (100% of both groups of

subjects were positive for HTLV-2 gag gene related DNA sequences).

Weaknesses of this study include:

• the use of a European case definition for CFS (6) which differed

substantially from that used in the United States

• failure to use a " hot start " PCR procedure such as that used by DeFreitas

et al.

• use of the DeFreitas et al. primers with reaction conditions

significantly different from those used by DeFreitas et al

• the DeFreitas et al. detection DNA probe failed to recognize the PCR

product obtained, suggesting nonspecificity of the PCR reaction.

The remaining three publications were from the same group of investigators

at the CDC (7,8,9). These studies which utilized the HTLV-2 specific DNA

primers used by DeFreitas et al. failed to detect HTLV-2 gag gene related

DNA sequences in either CFS patients or control subjects. This finding

stands in stark contrast to the observations of Gow et al. as described

above. Strengths of these three studies included: ‚ they were for the most

part performed in a blinded fashion ‚ they attempted to correlate their PCR

findings with a variety of epidemiological factors ‚ they sought a variety

of animal retroviruses in addition to HTLV-2.

Weaknesses of these three studies included:

• failure to use a " hot start " PCR procedure such as that used by DeFreitas

et al.

• use of the primers of DeFreitas et al. under experimental conditions

significantly different from those used by DeFreitas et al.

• the results of these studies are somewhat misleading in that the data

reported in all three papers apparently were obtained from a single

experiment involving 21 CFS patients and matched controls. No reference is

made to this fact in any of the three publications.

In conclusion, the results reported by DeFreitas et al. have not been

reproduced by other, independent laboratories. However, these subsequent

investigations differed substantially from the original DeFreitas et al.

studies in a number of ways, including:

• clinical definition of the CFS patients studied

• uniform failure to use a " hot start " procedure with the PCR assay

• use of experimental conditions for the PCR assay that significantly

differed from those used by DeFreitas et al

Specific Research Proposal

The goals of the proposed studies are:

• to use contemporary PCR techniques in an attempt to confirm the findings

of DeFreitas et al. with respect to the presence of HTLV-2 gag gene related

DNA sequences in the blood leukocytes of patients with CFS

• To correlate the presence of HTLV-2 gag gene related DNA sequences with

the presence of active infections with human herpesvirus six (HHV-6) or

Epstein-Barr virus (EBV)

Patients and Controls

The samples of peripheral blood leukocytes (PBL) to be used in these

studies are currently on hand in our laboratory. They have all been frozen

at -70oC for periods up to 3 years.

The samples will be of two types:

• The first set of PBL samples were obtained as part of a cross-sectional

study of CFS patients performed by our laboratory in collaboration with Drs.

Komaroff (Boston, Massachusetts) and (Incline

Village, Nevada). The samples consist of PBL from 20 patients with CFS and

from 20 matched healthy control individuals. Samples will be analyzed in a

blinded fashion.

• The second set of PBL samples will be PBL from CFS patients submitted to

our laboratory for virological analysis. These samples were submitted from

three physicians who specialize in the treatment of CFS patients: i.e. 10

samples from Dr. ph ( Wood Medical School; New

Brunswick, New Jersey), 10 samples from Dr. Cheney (Bald Head Island,

North Carolina) and 10 samples from Dr. Salvato (Houston, Texas).

• Thus the samples to be analyzed will be derived from 50 CFS patients and

20 healthy controls.

Detection of HTLV-2 gag Gene Related DNA Sequences

The PCR systems to be used in these studies can be summarized as follows:

• The original DNA primer system of DeFreitas et al. will be used with one

modification. In place of the radioactive probe procedure used in their

original studies, we will use a nested PCR system (Appendix 2) to provide

adequate sensitivity and specificity. Also, a pool of two downstream primers

differing in HTLV-2 subtype specific nucleotide sequences will be used in

order to allow equivalent detection of both types of the virus.

• A newly designed nested PCR system targeting a different region of the

HTLV-2 gag gene will be used in addition to the primer system of DeFreitas

et al. This PCR system was designed with contemporary nucleotide sequence

analysis software, which allows more precise prediction of binding affinity

and specificity, using the gag gene sequences from six strains of HTLV-2A

and five strains of HTLV-2B. This strategy allowed us to design primers that

will certainly detect both types of HTLV-2 with equivalent efficiency.

• DNA will be extracted and purified from patient and control samples of

blood leukocytes by means of a commercially obtained, widely used kit

(QIAmp DNA Mini Blood Kit; QIAGEN Inc; Valencia, California)(Appendix 3).

• Detection of PCR products in both PCR systems will be accomplished by

agarose gel electrophoresis with ethidium bromide staining.

Relationship Between the Presence of HTLV-2 gag Gene Related Sequences and

Active Lymphotropic Herpesvirus Infections

Numerous studies have established that herpesviruses and retroviruses can

intimately interact at the subcellular level (10,11,12). These interactions

range from cross-transactivation of each other's genes to actual integration

of the retrovirus genome into the much larger herpesvirus genome. There are,

in addition, data suggesting that active infections by HHV-6 and/or EBV may

be involved in the pathogenesis of CFS (K Knox and D Carrigan; data not

shown). Therefore, it will be of interest to correlate our findings

concerning HTLV and these two viruses in the studies proposed here.

Specifically, we will:

• correlate the detection of HTLV-2 gag gene DNA sequences with the results

obtained with the same sample in a rapid HHV-6 culture assay. All specimens

to be studied have already been analyzed by this rapid culture procedure

with numerous positive samples being identified.

• correlate the detection of HTLV-2 gag gene DNA sequences with the results

obtained with the same sample with a serum PCR procedure specific for EBV

DNA. Serum (or plasma) samples matched to the PBL specimens in these studies

are available in frozen form in our laboratory. The serum PCR procedure has

been successfully applied to samples from CFS patients (K Knox and D

Carrigan; data available upon request.

REFERENCES

1. DeFreitas E, Hilliard B, Cheney PR, Bell DS, Kiggundu E, Sankey D,

Wroblewska Z, Palladino M, Woodward JP AND Koprowski H. Retroviral

sequences related to human T?lymphotropic virus type II in patients with

chronic fatigue immune dysfunction syndrome. Proc Natl Acad Sci 1991;

88:2922?2926.

2. Defreitas E and Hilliard B. Method and compositions for diagnosing and

treating chronic fatigue immunodysfunction syndrome. International Patent

Application Number PCT/US91/06238; April, 1992.

3. Holmes GP, Kaplan JE, Gantz NM et al. Chronic fatigue syndrome: a

working case definition. Ann Intern Med 1988; 108:387-389.

4. Gunn WJ, Komaroff AL, Bell DS et al. Inability of retroviral tests to

identify persons with chronic fatigue syndrome, 1992. Morb Mortal Wkly

Rep.1993;42:189?190.

5. Gow JW, Simpson K, Schliephake A, Behan WM, on LJ, Cavanagh H,

Rethwilm A and Behan PO. Search for retrovirus in the chronic fatigue

syndrome. J Clin Pathol. 1992; 45:1058?1061.

6. Behan PO and Bakheit AMO. Clinical spectrum of postviral fatigue

syndrome. Brit Med Bull 1991; 47:793-808.

7. Khan AS, Heneine WM, Chapman LE, HE Jr, Woods TC, Folks TM and

Schonberger LB. AssessmSent of a retrovirus sequence and other possible

risk factors for the chronic fatigue syndrome in adults. Ann Intern Med.

1993;118:241?245.

8. Folks TM, Heneine W, Khan A, Woods T, Chapman L and Schonberger L.

Investigation of retroviral involvement in chronic fatigue syndrome. Ciba

Found Symp. 1993; 173:160?166.

9. Heneine W, Woods TC, Sinha SD, Khan AS, Chapman LE, Schonberger LB,

Folks TM. Lack of evidence for infection with known human and animal

retroviruses in patients with chronic fatigue syndrome. Clin Infect Dis.

1994; 18 Suppl 1:S121?S125.

10. Isfort R, , D, Kost R, Witter R and Kung HJ. Retrovirus insertion

into herpesvirus in vitro and in vivo. Proc Nat Acad Sci 1992; 89:991-995.

11. Isfort RJ, Witter R and Kung HJ. retrovirus insertion into

herpesviruses. Trends Microbiol 1994; 2:174-177.

12. D, Liu JL, D et al. Avian leukemias and lymphomas:

Interplay between retroviruses and herpesviruses. Leukemia 1997; 11 (suppl

3):176-178.

© 2001, 2002 Institute for Viral Pathogenesis. All rights reserved.

----------------------------------------------------------------------

This relates to their references on retroviruses in herpesviruses

http://www.mcw.edu/microbiology/seg.html

Grossberg, S.E.,

Our current research deals with the molecular, immunological, and biological

characterization of a new human RNA virus that replicates in human

B-lymphoblastoid cells. The virus, designated JHKV, is an enveloped,

relatively fragile particle containing RNA, reverse transcriptase, and

prominent, knobbed glycoprotein projections. Although the JHK virus

resembles a retrovirus (but 35% smaller than most other retroviruses), it is

clearly not like any of the known human retroviruses (human immunodeficiency

virus or human T-lymphotropic virus), as determined either by polymerase

chain reactions or electron microscopy. The JHK-3 B-lymphoblastoid cell line

that constitutively produces the JHK virus (and the Epstein-Barr virus as

well) has the antigenic markers of immature B-lymphocytes by flow cytometry.

cDNA libraries produced by reverse transcription of JHK viral RNA have been

constructed and are being analyzed; the sequences determined of the many

clones produced have so far revealed no significant homology with known

viruses. Polyclonal antibodies and certain monoclonal antibodies we have

produced react with JHKV by enzyme-linked immunosorbant assay, Western

immunoblot, and immunogold binding, like sera from certain patients. The

possible etiological role of the JHK virus in diseases that may involve

B-lymphocytes, such as leukemia, lymphoma, chronic fatigue syndrome or

immuno-dysfunctional states, remains to be determined. The goals of current

experimental studies are to (i) clone, sequence, and map the viral genome;

(ii) characterize monoclonal antibodies to the virus; (iii) identify viral

antigens; and (iv) develop methods to detect JHKV infection, using nucleic

acid probes for hybridization and primers for the polymerase chain reaction.

Grossberg, S.E., and Kawade, Y.: The Expression of Potency of Neutralizing

Antibodies for Interferons and other Cytokines. Biotherapy 10:93-98, 1997.

Abstract

Grossberg, S.E., Kushnaryov, V.M., Cashdollar, L.W., Raisch, K.P., ,

G., and Sun, H-Y.: A Human B-lymphoblastoid Cell Line Constitutively

Producing Epstein-Barr Herpesvirus and JHK Retrovirus. Research in Virology

148:191-206, 1997.

Abstract

, J.L., and Grossberg, S.E.: The Effects of Interferon-alpha on the

Production and Action of Other Cytokines, Seminars in Oncology, 25:23-29,

1998.

Abstract

Raisch, K.P., Kushnaryov, V.M., Grossberg, S.E., and Cashdollar, L.W.:

Constitutive Production of a Murine Retrovirus in the Human B-Lymphoblastoid

Cell Line DG-75. Virology 250: 135-139, 1998.

Abstract

Pungor E Jr, Files JG, Gabe JD, Do LT, Foley WP, Gray JL, JW, Nestaas

E, JL, Grossberg SE. A novel bioassay for the determination of

neutralizing antibodies to IFN-beta1b. J Interferon Cytokine Res.

18(12):1025-30,1998. Abstract

Golgher, R.R., Redlich, P.N., Delvia, O.S.B., Totti, D.O. and Grossberg,

S.E.: Quantitative Liquid-Phase Chemiluminescence ELISA: Detection of

Subtle Epitope Differences in Human Interferon-beta. J Interferon Cytokine

Res. 19: 995-998, 1999. Abstract

Osorio JE, Grossberg SE, Palmenberg AC. Characterization of genetically

engineered mengoviruses in mice. Viral Immunol. 13(1):27-35, 2000. Abstract

_________________________________________________________________

Get your FREE download of MSN Explorer at http://explorer.msn.com/intl.asp.