Reduced Glutathione and Satratoxins (runningantelope)

Discussion in 'Fibromyalgia Main Forum' started by Slayadragon, Oct 10, 2009.

  1. Slayadragon

    Slayadragon New Member

    I was hoping to get thoughts from Rich van K and Bill (RunningAntelope), but others' comments are welcome.

    I was reading some discussion on the board today that reminded me of the following dissertation related to satratoxins (poisons made by stachybotrys).

    The fact that it conceivably could be related to XMRV makes me feel like it's especially topical.

    Bill and Rich, what do you think?

    Thanks, Lisa

    *

    http://etd.lib.ttu.edu/theses/available/etd-05252005-163223/unrestricted/Karunasena_Enusha_Diss.pdf

    OR

    http://etd.lib.ttu.edu/theses/available/etd-05252005-163223/

    *

    During an inflammatory response, and early apoptotic events cells are
    under oxidative stress, which leads to the production of lipid radicals and lipid
    peroxidation. Previous studies have demonstrated that lipid radicals are able to
    inhibit anti-apoptotic genes which allow a cell to enter into apoptosis. In these
    experiments, the presence of oxidative stress was evaluated by measuring lipid
    hyperperoxides (LOOH) and reduced glutathione (GSH) levels according to
    established methods [21, 22].

    Established methods were utilized to measure oxidative stress conditions
    to determine the concentration of GSH [21]. In a normal cell, GSH is unable to
    cross the nuclear membrane, but during oxidative stress the oxidized form of
    GSH, oxidized glutathione (GSSG), is able to cross this structure. GSSG is able
    to decrease the binding of p60/p65 complex of NF- ?B to DNA, which reduces
    the pro-inflammatory cascade that is activated by NF- ?B. The intracellular GSH
    levels were measured from cell homogenates [21]. The reaction of GSH with 5, 5’
    dithiobis and sulfhydryl compounds leads to a color change, producing a yellow
    pigment. The samples were read spectrophotometrically at 412nm (Sigma
    Chemical Co., St Louis, MO). Results were compared to a standard curve using
    GSH in nanomoles per milligram of protein. Protein concentrations for the cell
    homogenate fractions were determined using a BCA Protein Assay Kit (Pierce,
    Rockford, IL).
    47

    Lipid hydroperoxides (LOOH) were evaluated in cell homogenate fractions
    using previously established methods [21, 22]. This assay is able to directly
    measure the LOOH concentration Samples were mixed with SDS-acetate buffer,
    pH3.5, and aqueous solution of thiobarbituric acid. The reaction mixture was
    heated at 95?C for 90 minutes. After cooling, the red-colored complex was
    extracted with n-butanol-pyridine [21, 22]. The data were measured
    spectrophotometrically at 532 nm, and compared to a standard curve [21, 22].
    Results were expressed in nanomoles of LOOH per microgram of cells [21, 22].
    Statistical analysis (?= 0.05) was performed using Sigma Stat, a statistical
    software program designed by Jandel (SPSS), to analyze the data using one-
    way analysis of variance (ANOVA) of each experimental group with the controls.
    Normality of the data was determined using a Kolmogorov-Smirnov normality
    test. If data did not meet normality, a non-parametric Kruskal-Wallis ANOVA
    would have been applied. If data were normal, and ANOVA demonstrated
    significance, a post hoc test, Tukey test (a modified t test), was used to make
    multiple comparisons to determine which experimental groups demonstrated
    significance compared to the controls and between experimental groups.
    Results were graphed using Sigma Plot, a graphical program designed by Jandel
    (SPSS).

    48
    RESULTS

    In these experiments HBCEC were evaluated for the expression of
    inflammatory and apoptotic events from the exposure of satratoxin H, LPS, and
    H202. Negative controls were exposed to sterile, pyrogen-free water, and positive
    controls were exposed to 50EU/ml LPS and 250µM H202 in a volume of 20µl.
    These cells were incubated for a period of 18h at 37?C with 5% CO2 in 6 well
    plates and unsiliconized coverslips. The evaluation of LDH levels demonstrated
    significant (P<0.05) cytotoxic events in cells exposed to 1000ng/ml SH and
    additive conditions (10ng/ml + LPS and 10ng/ml + H2O2). These results can be
    seen in Figure 2.

    Cells were later evaluated for the expression of adhesion molecule
    receptors (ICAM, VCAM, P/E selectin) expressed in the event of inflammation.
    Figures 3, 5, and 7 demonstrate immunofluorescent results from the expression
    of ICAM, VCAM and P/E selectin. Cells that expressed the adhesion molecules
    receptors on the surface expressed were bound by Ab conjugated to the green
    fluorescent stain FITC. A live video-camera attached to the microscope was used
    to photograph three sections of each slide. A total of 6 slides per an experimental
    group were evaluated. Pictures of the cells were further evaluated to quantitate
    the density of fluorescence using Scan Analysis Software (BioSoft, Cambridge,
    UK). Figures 4, 6, and 8 demonstrate the degree of fluorescence produced by
    cells. The total area beneath a peak was used to determine the intensity of
    49
    receptor expression for each sample group.

    Figure 4 demonstrates a significant
    increase (P< 0.05) in the expression of ICAM on cells exposed to satratoxin H
    100ng/ml, 1000ng/ml, and 10ng/ml + H202. VCAM expression was significantly
    greater (P<0.05) than control cells when exposed to 100ng/ml SH, LPS, H202,
    10ng/ml + LPS, and 10ng/ml + H202. A significant expression (P< 0.05) of P/E
    selectin was detected in HBCEC cells exposed to 100ng/ml, 1000ng/ml, LPS,
    H202, 10ng/ml + LPS, and 10ng/ml + H202.

    Further evidence of damage to the BBB, is EC cell shrinkage which leads
    to greater permeability across the BBB. To evaluate whether satratoxin H is able
    to induce HBCEC shrinkage, a monolayer of cells grown on 0.4µm polycarbonate
    membranes in 24 well plates were exposed to 125I- albumin and monitored every
    15min for the diffusion rate of albumin across the monolayer. There was a
    significantly greater rate (P< 0.05) of diffusion across cells exposed to 100ng,
    and 1000ng/ml of SH. A significant (P< 0.05) additive effect was observed in cells
    exposed to 10ng/ml + LPS and 10ng/ml + H202. These results are seen in Figure


    Apoptotic events were observed in addition to inflammation. An annexin V
    apoptotic detection assay (Sigma, St. Louis, MO) was utilized to evaluate
    apoptosis. In the event of early apoptosis, phosphatidylserine (PS) expressed on
    the inner cell membrane is flipped to the outer surface of the cell membrane as
    an indication of apoptosis. In the detection assay, secondary Ab conjugated to
    FITC (green) binds to PE on the surface of cells in the event of apoptosis. Late
    50
    stages of apoptosis consist of chromatin fragmentation and permeability of the
    nuclear membrane. In the event of late stages of apoptosis, propidium iodide
    (red) binds to damage chromatin material. These events are observed in Figure
    10.

    Compared to the negative control cells that received water, cells exposed to
    100ng/ml SH, and 1000ng/ml SH, and LPS demonstrated early and late stages
    of apoptosis, whereas the control cells did not have a red stain in the nucleus of
    the cell. To further evaluate apoptosis, cytochrome C levels from cell extracts
    were evaluated using an ELISA method. These results demonstrated that a
    significantly increased amount (P< 0.05) of cytochrome C was released from
    cells exposed to 10ng/ml, 100ng/ml, LPS, 10ng/ml + LPS, and 10ng/ml + H202.
    These results can be seen in figure 11.

    An additional indicator of apoptosis is oxidative stress. In the event of
    oxidative stress, glutathione (GSH) acts as a reducing agent against reactive
    oxygen species (ROS) such as lipid radicals and peroxides.

    However, if GSH
    levels in a cell are insufficient to compensate for the degree of oxidative stress,
    both apoptotic and inflammatory pathways are further activated.

    To determine
    whether mycotoxins increased oxidative stress levels in HBCEC, a quantitative
    method was used to determine the levels of GSH present in cell extracts
    exposed to various experimental conditions.

    The results demonstrated a
    significant decrease (P> 0.05) in the concentration of GSH (µg/ml) in cells
    exposed to 100ng/ml SH, 1000ng/ml SH, LPS, H202, 10ng/ml + LPS, 10ng/ml +
    H202. These results are seen in Figure 12. The production of lipid peroxidation,
    51
    further demonstrates the degree of oxidative stress induced on HBCECs.

    Results
    from the thiobarbituric acid assay (T-BARS) demonstrated that there was a
    significant increase (P>0.05) in lipid peroxidation when cells were exposed LPS,
    H2O2, moderate concentrations of SH (100ng/ml and 1000ng/ml), and additive
    conditions (10ng/ml + LPS and 10ng/ml + H2O2). These results can be seen in
    Figure 13.
    52


    CONCLUSIONS
    Results from the adhesion mo le receptor expression on HBCEC
    demonstrate that satratoxin H levels of 100ng/ml and 1000ng/ml are able to
    induce inflammatory pathway activation alone.

    Additive effects are demonstrated
    with very low concentrations of SH, such as 10ng/ml in the presence of
    inflammatory agents such as LPS and H202.

    Similar concentrations of the
    mycotoxin are able to induce apoptotic pathways leading to the activation of early
    stages of apoptosis in the presence of 100ng/ml SH, however evidence of late
    stages of apoptosis are observed with 1000ng/ml and 10ng/ml + LPS or 10ng/ml
    H202.

    These results demonstrate the ability of satratoxins to induce apoptotic
    pathways at the same concentrations that inflammatory pathways are being
    activated.

    This suggests that low levels of inflammation and apoptotic events can
    be induced in the presence of moderate levels of SH, and low levels of SH are
    able to induce similar events in the presence of other inflammatory agents and
    oxidative stress conditions, as demonstrated by the levels of GSH and
    cytochrome C in cell extracts.

    In addition, the ability of the mycotoxins to induce
    cell shrinkage at moderate to low levels of SH demonstrate the potential ability of
    these agents to compromise the integrity of the BBB which could lead to further
    neurological damage from mycotoxins or other harmful agents.

    The presence of
    lipid peroxidation in cells exposed to moderate concentrations of SH and additive
    conditions, further demonstrates the ability of the mycotoxins to amplify cellular
    65
    damage through the indirect production of lipid radicals and other ROS.

    The
    results further suggest that low to moderate levels of SH are able to induce
    inflammatory and apoptotic pathways that amplify the cellular damage by the
    continuous activation of these biological pathways.


    *

    During an inflammatory response and early apoptotic events, cells are
    under oxidative stress, which leads to the production of lipid radicals and lipid
    peroxidation.

    Previous studies have demonstrated that lipid radicals are able to
    inhibit anti-apoptotic genes which allow a cell to enter into apoptosis. In these
    experiments, the presence of oxidative stress was evaluated by measuring lipid
    hyperperoxides (LOOH) and reduced glutathione (GSH) levels according to
    established methods [13-16].

    In a normal cell GSH is unable to cross the nuclear membrane, but during
    oxidative stress the oxidized form of GSH, oxidized glutathione (GSSG), is able
    to cross this structure. GSSG is able to decrease the binding of p60/p65 complex
    of NF- ?B to DNA, which reduces the pro-inflammatory cascade that is activated
    by NF- ?B. The intracellular GSH levels were measured from cell homogenates
    [15]. The reaction of GSH with 5, 5’ dithiobis and sulfhydryl compounds leads to
    78
    a color change, producing a yellow pigment.

    The samples were read
    spectrophotometrically at 412nm (Sigma Chemical Co., St Louis, MO). Results
    were compared to a standard curve using GSH in nanomoles per milligram of
    protein. Protein concentrations for the cell homogenate fractions were
    determined using a BCA Protein Assay Kit (Pierce, Rockford, IL).

    Lipid hydroperoxides (LOOH) were evaluated in cell homogenate fractions
    with a previously established method [13-16]. This assay is able to directly
    measure the LOOH concentration Samples were mixed with SDS-acetate buffer,
    pH3.5, and aqueous solution of thiobarbituric acid. The reaction mixture was
    heated at 95?C for 90 minutes. After cooling, the red-colored complex was
    extracted with n-butanol-pyridine [13-16]. The data were measured
    spectrophotometrically at 532 nm, and compared to a standard curve [13-16].
    Results were expressed in nanomoles of LOOH per microgram of cells [13-16].
    Statistical analysis (?= 0.05) was performed using Sigma Stat, a statistical
    software program designed by Jandel (SPSS), to analyze the data using one-
    way analysis of variance (ANOVA) of each experimental group with the controls.
    Normality of the data was determined using a Kolmogorov-Smirnov normality
    test. If data did not meet normality, a non-parametric Kruskal-Wallis ANOVA
    would have been applied. If data were normal, and ANOVA demonstrated
    significance, a post hoc test, Tukey test (a modified t test), was used to make
    multiple comparisons to determine which experimental groups demonstrated
    significance compared to the controls and between experimental groups.
    79
    Results were graphed using Sigma Plot, a graphical program designed by Jandel
    (SPSS).
    RESULTS

    In these experiments astrocytes were evaluated for the expression of
    inflammatory and apoptotic events from the exposure of satratoxin H, LPS, and
    H202. Negative controls were exposed to sterile, pyrogen-free water, and positive
    controls were exposed to 50EU/ml LPS and 250µM H202 in a volume of 20µl.
    These cells were incubated for a period of 18h at 37?C with 5% CO2 in 6 well
    plates and unsiliconized coverslips. Cytotoxicity results were determined by LDH
    levels measured from cell media. The results demonstrate significant (P<0.05)
    cytotoxicity in cell exposed to 1000ng/ml SH and additive conditions (10ng/ml SH
    + LPS, and 10ng/ml + H202). These results are shown in figure 14.
    Cells were later evaluated for the expression of adhesion molecule
    receptors (ICAM, VCAM, P/E selectin) expressed in the event of inflammation.
    Figures 15, 17, and 19 demonstrate immunofluorescent results from the
    expression of ICAM, VCAM and P/E selectin. Cells that expressed the adhesion
    molecules receptors on the surface were bound by Ab conjugated to the green
    fluorescent stain FITC. A live video-camera attached to the microscope was used
    `to photograph three sections of each slide. A total of 6 slides per an experimental
    group were evaluated. Pictures of the cells were further evaluated to quantitate
    the density of fluorescence using Scan Analysis Software (BioSoft, Cambridge,
    UK). Figures 16, 18, and 20 show levels of fluorescence produced by cells. The
    80
    total area beneath a peak was used to determine the intensity of receptor
    expression for each sample group. These results demonstrate that there is a
    significant increase (P<0.05) in the expression of ICAM, VCAM, and P/E selectin
    under additive conditions (100ng/ml SH + LPS and 100ng/ml SH + H202).

    The role of astrocytes in neural tissue is to produce protective
    inflammatory compounds in the event of disease. Immune cells are able to
    specifically amplify immune responses by the activation of transcription factors
    such as NF-?B. The activation of NF-?B leads to the nuclear translocation of the
    p60/p65 subunit. The binding of the subunit to DNA leads to the transcription of
    cytokines and chemokines that further amplify the inflammatory process.

    Using
    an ELISA method, the concentration of NF-?B was quantitated as a measure of
    inflammation induced by SH. Results demonstrate a significant increase (P<0.05)
    in the expression of NF-?B in cells exposed to 100ng/ml SH, 1000ng/ml SH, LPS,
    100ng/ml SH + LPS, and 100ng/ml + H202. These results can be seen in figure
    21.

    Apoptotic events were observed in addition to inflammation. An annexin V
    apoptotic detection assay (Sigma Aldrich) was utilized to evaluate apoptosis.

    In
    the event of early apoptosis, phosphatidylserine (PS) expressed on the inner cell
    membrane is flipped to the outer surface of the cell membrane as an indication of
    apoptosis. In the detection assay, secondary Ab conjugated to FITC (green)
    binds to PE on the surface of cells in the event of apoptosis.

    Late stages of
    apoptosis consist of chromatin fragmentation and permeability of the nuclear
    81
    membrane. In the event of late stages of apoptosis, propidium iodide (red) binds
    to damaged chromatin material. These events can be seen in figure 22.

    Compared to the negative control cells that received water, cells exposed to
    100ng/ml SH, and 1000ng/ml SH, and LPS demonstrated early and late stages
    of apoptosis, whereas the control cells did not have a red stain in the nucleus of
    the cell. To further evaluate apoptosis, cytochrome C levels from cell extracts
    were evaluated using an ELISA method. These results demonstrated that a
    significant increase (P< 0.05) in the amount of cytochrome C released from cells
    occurred when they were exposed to 100ng/ml SH, 1000ng/ml SH, LPS, 10ng/ml
    + LPS, and 10ng/ml + H202. Figure 23 shows these results.

    An additional indicator of apoptosis is oxidative stress. In the event of
    oxidative stress, glutathione (GSH) acts as a reducing agent against reactive
    oxygen species (ROS) such as lipid radicals and peroxides.

    However, if GSH
    levels in a cell are insufficient to compensate for the degree of oxidative stress,
    both apoptotic and inflammatory pathways are further activated.

    To determine
    whether mycotoxins increased oxidative stress levels in astrocytes, an ELISA
    method was used to quantitate the levels of GSH present in cell extracts exposed
    to various experimental conditions. The results demonstrated a significant
    decrease (P> 0.05) in the concentration of GSH (µg/ml) occurred in cells
    exposed to 10ng/ml -1000ng/ml, LPS, H202, 100ng/ml + LPS, 100ng/ml + H202
    (Figure 24).
    82

    The presence of ROS, such as lipid peroxides was determined by a T-
    BARS assay to quantitate the degree of lipid peroxidation due to the
    experimental conditions evaluated. These results demonstrated that, at moderate
    concentrations of SH (100ng/ml and 1000ng/ml), lipid peroxidation was
    significantly higher (P> 0.05) compared to control conditions. Additive conditions,
    such as 100ng/ml SH + LPS and 100ng/ml SH + H202 produced significant
    (P>0.05) levels of lipid radicals. These results can be seen in Figure 25.

    *

    CONCLUSIONS

    These studies demonstrate that mycotoxins at moderate concentrations
    and under additive conditions are able to produce cytotoxic events.

    These results
    demonstrate that direct exposure of astrocytes to satratoxin H at low to moderate
    concentrations alone do not produce a strong inflammatory response as
    evidenced by no significant increase in ICAM, VCAM, and P/E selectin as well
    as NF-?B expression.

    However, these results do demonstrate an additive effect
    in the expression of inflammatory events with a moderate dose (100ng/ml) of
    satratoxin H in the presence of other inflammatory compounds (LPS) or under
    oxidative stress conditions.

    Early and late apoptotic events are evidenced with moderate levels of
    satratoxin H (100ng/ml and 1000ng/ml), as demonstrated by the Annexin V
    assay for apoptosis. In addition, the evaluation of cytochrome C and GSH levels
    demonstrate that moderate concentrations of SH alone and under additive
    conditions of pro-inflammatory compounds and oxidative stress, produce
    significantly higher levels (P>0.05) of apoptosis in astrocytes.

    These results demonstrate that moderate levels of mycotoxins are able to
    activate inflammatory pathways in astrocytes under additive conditions, which
    could lead to increased expression of inflammatory compounds by astrocytes.
    This would increase the production of ROS, such as lipid radicals through lipid
    peroxidation, under oxidative stress conditions, which would lead to cell damage.
    96

    Further evaluation of these data also demonstrates that astrocytes significantly
    increase (P> 0.05) the expression of apoptotic events, alone and under additive
    conditions.

    The production of ROS can further cause cell damage under
    conditions of continuous inflammatory and apoptotic pathway activation.

    These results further suggest that the production of pro-inflammatory and
    apoptotic compounds by astrocytes, when released in the environment of neural
    tissues, could activate HBCEC and neurons leading to inflammation and
    apoptosis.

    These studies propose that under SBS conditions, individuals
    exposed to satratoxin H and microbial organisms in the environment over a
    prolonged period could have increased sensitivity to these agents, leading to
    neural damage.


    *

    In a normal cell GSH is unable to cross the nuclear membrane, but during
    oxidative stress the oxidized form of GSH, oxidized glutathione (GSSG), is able
    to cross this structure. GSSG is able to decrease the binding of p60/p65 complex
    of NF- ?B to DNA, which reduces the pro-inflammatory cascade that is activated
    by NF- ?B. The intracellular GSH levels were measured from cell homogenates
    [5]. The reaction of GSH with 5, 5’ dithiobis and sulfhydryl compounds leads to a
    color change, producing a yellow pigment. The samples were read
    spectrophotometrically at 412nm (Sigma Chemical Co., St Louis, MO). Results
    were compared to a standard curve using GSH in nanomoles per milligram of
    103
    protein. Protein concentrations for the cell homogenate fractions were
    determined using a BCA Protein Assay Kit (Pierce, Rockford, IL).
    During an inflammatory response, cells are under oxidative stress, which
    leads to the production of lipid radicals and lipid peroxidation. Previous studies
    have demonstrated that lipid radicals are able to inhibit anti-apoptotic genes
    which allow a cell to enter into apoptosis. In these experiments, the presence of
    oxidative stress was evaluated by measuring lipid hyperperoxides (LOOH) and
    reduced glutathione (GSH) levels according to established methods [5-8].
    LOOH were evaluated in cell homogenate fractions with a
    thiobarbaturic acid assay (T-BARS). This assay is able to directly measure the
    LOOH concentration. Samples were mixed with SDS-acetate buffer, pH3.5, and
    aqueous solution of thiobarbituric acid. The reaction mixture was heated at 95?C
    for 90 minutes [5-8]. After cooling, the red-colored complex was extracted with n-
    butanol-pyridine. The data were measured spectrophotometrically at 500nm, and
    compared to a standard curve [5-8]. Results were expressed in nanomoles of
    LOOH per microgram protein [5-8].

    Statistical analysis (?= 0.05) was performed using Sigma Stat, a statistical
    software program designed by Jandel (SPSS), to analyze the data using one-
    way analysis of variance (ANOVA) of each experimental group with the controls.
    Normality of the data was determined using a Kolmogorov-Smirnov normality
    test. If data did not meet normality, a non-parametric Kruskal-Wallis ANOVA
    would have been applied. If data were normal, and ANOVA demonstrated
    104
    significance, a post hoc test, Tukey test (a modified t test), was used to make
    multiple comparisons to determine which experimental groups demonstrated
    significance compared to the controls and between experimental groups.
    Results were graphed using Sigma Plot, a graphical program designed by Jandel
    (SPSS).

    RESULTS
    Results from the LDH assay demonstrate a significant increase (P<0.05)
    in the release of LDH by neurons exposed to all of the experimental conditions
    except LPS, H2O2, and 10ng/ml SH.

    Moderate concentrations of SH and additive
    conditions demonstrated cytotoxic events as measured by the concentration of
    LDH released into the media compared to control conditions. These results can
    be seen in Figure 26.

    Cytochrome C levels from cell extracts were evaluated using an ELISA method.
    These results demonstrated that a significant amount (P< 0.05) of cytochrome C
    was released from cells exposed to all of the experimental conditions compared
    to control conditions (H20). These data are presented in Figure 27.

    An additional indicator of apoptosis is oxidative stress. In the event of
    oxidative stress, glutathione (GSH) acts as a reducing agent against reactive
    oxygen species (ROS) such as lipid radicals and peroxides. However, if GSH
    levels in a cell are insufficient to compensate for the degree of oxidative stress,
    both apoptotic and inflammatory pathways are further activated.

    To determine
    whether mycotoxins increased oxidative stress levels in astrocytes, an ELISA
    105
    method was used to quantitate the levels of GSH present in cell extracts exposed
    to various experimental conditions. These data demonstrated that compared to
    control conditions there was significantly less (P>0.05) GSH from cell extracts
    exposed to the experimental conditions previously described. These data are
    presented in Figure 28.

    During the event of oxidative stress, ROS such as lipid radicals are
    produced due to lipid peroxidation. To quantitate the degree of lipid peroxidation,
    a T-BARS assay was conducted to measure the concentration produced by cells
    exposed to the previously described experimental conditions.

    The data
    demonstrated that neurons exposed to all of the experimental conditions
    produced significant levels (P<0.05) of lipid peroxidation compared to the control
    conditions. These results can be seen in Figure 29.


    [This Message was Edited on 10/11/2009]
  2. richvank

    richvank New Member

    Hi, Lisa.

    I can't vouch for the validity of these detailed measurements, since I don't have experience in this type of research. However, it appears that the conclusion from this work was that satratoxin H, one of the toxins produced by Stachybotros types of molds, produces oxidative stress and depletion of glutathione in the types of cells that were studied.

    If this is true, I think it could provide a link between mold illness and CFS.

    If a person has both the HLA genomic susceptibility to mold illness, and also the genomic susceptibility to developing a partial methylation cycle block, then I think that exposure to a mold toxin of this type could produce both mold illness and chronic fatigue syndrome. I think that both would have to be treated for the person to recover their health. There are people who get mold illness, but not CFS, and I think those are the ones Dr. Shoemaker has been able to help with avoidance and cholestyramine. I think that the ones he hasn't been able to bring to recovery may be stuck in CFS, because of their inherited genomic predispostion. I'm hopeful that the combined treatment will help them, but that study is still in the future.

    As you may know, this appears to me to be the link between Lyme disease and CFS, also. Borrelia burgdorferi bacteria have been found to lower the glutathione level in their hosts. If a person is genomically predisposed to develop a methylation cycle block when glutathione is lowered sufficiently, I think they will develop CFS along with their Lyme disease, and again, both will need to be treated for them to recover. I think this may explain the socalled "post-Lyme disease syndrome." Some of the Lyme doctors seem to be trying a combined treatment now. Two of them mentioned the methylation work in the new book on insights into the treatment of Lyme disease, by Connie Strasheim. I had a good talk with Dr. Shor at the IACFS/ME conference, and he believes in a link between Lyme and CFS, too. There are people who get Lyme disease and recover from it after antibiotic treatment. These may be the ones who do not have a predisposition toward the methylation cycle block.

    Just for completeness, let me say that I think that multiple chemical sensitivity can arise when glutathione becomes depleted in the sustentacular (suppporting) cells in the olfactory epithelium in the nose. I think that's the link between CFS and MCS.

    I don't yet have a hypothesis for the link between CFS and fibromyalgia. My friend David Gregg things fibro has a viral basis, from a virus that lives in neurons, but so far there isn't a lot of evidence about that. Maybe this new XMRV is a candidate, I don't know.

    Best regards,

    Rich
  3. Slayadragon

    Slayadragon New Member

    Hi, Rich.

    Thanks so much for your comments on this!

    It seems to me that the decreased effect of the satratoxins on reduced glutathione is pretty severe. This study was supposed to be duplicating low levels of poisonous exposure too.

    Do you have a sense of how this effect might compare to the other stress factors that you previously have cited in your models?

    Insofar as this is a really strong effect, would it help to resolve the question of why it is that CFS patients have SO much oxidative stress that they need to work so hard to reverse (e.g. with your supplements and Paul Cheney's many suggestions)?

    Both Erik and I benefited from measures such as Dr. Cheney's suggestions prior to mold avoidance. Following a few months of just moderate avoidance, and thereafter, we've been able to break ALL of Dr. Cheney's rules without negative effect.

    (I wish that continuing to follow these rules would decrease mold reactivity. I'd much rather follow the rules than avoid mold. But no, it doesn't work that way. Again, that makes me think that the mold has a really significant effect compared to minor effects from all these other stressors.)

    I'm still taking the FolaPro and B12. I don't know if they're still helping. But I do know that in the past if I stopped and re-started, I'd get a significant detox response for a day or two upon re-starting. That doesn't happen now.

    So maybe my block has been fixed. If so, it has to be as a result of the mold avoidance. Other than that, the only thing I've been doing to support my health is taking Vitamin C (10-15 g per day) and cholestyramine.

    On another note, your comment on MCS was inspiring. I wrote you a separate post on that topic.

    Best, Lisa
    [This Message was Edited on 10/11/2009]
  4. RunningAntelope

    RunningAntelope New Member

    "An additional indicator of apoptosis is oxidative stress. In the event of
    oxidative stress, glutathione (GSH) acts as a reducing agent against reactive
    oxygen species (ROS) such as lipid radicals and peroxides.

    However, if GSH
    levels in a cell are insufficient to compensate for the degree of oxidative stress,
    both apoptotic and inflammatory pathways are further activated."

    I think Mssrs. Cheney and Rich V would both concur. However, cell apoptosis is induced by many variables, not just insufficient glutathione, but it is certainly perpetuate and exacerbated in a state of oxidative stress. As previously noted, the body utilizes many enzyme systems to address reactive oxygen species, not the least of which are superoxide or peryoxynitrite (lethal). Read up on iNos when you get a chance. I think Rich reaches some good conclusions, and this could be "the" mold catalyst connection, but if the body lacks sufficient catalase or superoxide dismutase for example, because, say a retrovirus like HIV or XMRV comes along with complementary gene sequences capable of "knocking them out," the resultant superoxide thrown off by P450/NADPH decoupling could interact with mycotoxins and further shift the redox state. All of these theories overlap to some degree, along with Marty Pall's.

    This is what struck me about the article:

    "These results
    demonstrate that direct exposure of astrocytes to satratoxin H at low to moderate
    concentrations alone do not produce a strong inflammatory response as
    evidenced by no significant increase in ICAM, VCAM, and P/E selectin as well
    as NF-?B expression.

    However, these results do demonstrate an additive effect
    in the expression of inflammatory events with a moderate dose (100ng/ml) of
    satratoxin H in the presence of other inflammatory compounds (LPS) or under
    oxidative stress conditions."

    "In a normal cell GSH is unable to cross the nuclear membrane, but during
    oxidative stress the oxidized form of GSH, oxidized glutathione (GSSG), is able
    to cross this structure. GSSG is able to decrease the binding of p60/p65 complex
    of NF- ?B to DNA, which reduces the pro-inflammatory cascade that is activated
    by NF- ?B"

    If the new XMRV retroviral theory is even remotely correct, as I believe it to be, the NF-Kappa-B region is certainly in play for exploitation by the virus to produce its replicative progeny. This virus would ostensibly be capable of knocking out the body's enzymes capable of dealing with a state of ROS, but that needs to be firmly established before conclusions can be drawn. Admittedly, I need to do more research. Rich's research is interesting, and his results fruitful, but the change in the ratio of GSH/GSSG seemed to be insignificant. This research would seem to suggest that the mold is "piling on," but not able to significantly induce NF-kappa-B activation, which is what my "hunch" is. It's the retrovirus that is capable of that activation, and a state of perpetual ROS instability is modulated by these other stressors. But the predisposition Rich spoke of probably comes into play.

    I need to look at this some more. Curiously, in the literature I've seen, the astrocytes could be greatly effected by stem cells in a positive manner.
  5. Slayadragon

    Slayadragon New Member

    Hi, Bill.

    Certainly mold even in enormous quantities doesn’t do to normal people what it does to me in absurdly miniscule quantities.

    The idea that a virus needs to be present in order for any of the effects to occur thus makes sense to me.

    I think that what Rich is saying is that if you can get GSH high enough, you can keep the virus in check.

    But with the virus already being present and active, mold exposures (perhaps even a little bit of mold exposures) will destroy the GSH.

    Some of the GSH can be put back (through Rich’s or Cheney’s suggestions), but possibly not all of it.

    If you can get mold exposures down to zero, the GSH should come back up.

    This will keep the virus from growing.

    OK. That’s where I am now. What do I do next?

    Now that (presumably) the virus is mostly under control, what can I do to keep it that way even when I get some new mold exposures?

    If I were to take an antiviral now and get the levels really down, and then periodically do so into the future, would that be enough to keep the virus in check?


    Because if the virus weren’t expressing itself (even if it were present), I could be around large quantities of mold without being affected by it.

    Then I would be wholly well and (at least in my case) the puzzle would be solved.


    BTW, LPS (lipopolysaccharides) are provided by various bacteria (probably including Lyme, mycoplasma and cpn).

    The idea that Lyme (and/or these other bugs) exacerbates the effects of the mold is consistent with my own experience. When I was taking doxy, the extent to which I experienced die-off symptoms from day to day was related to my current mold exposures.

    It’s also consistent with Dr. Cheney’s and Peterson’s observations that they don’t see “pure” Lyme cases.

    It seems from this that Lyme (and those other bacteria) by itself is no big deal, that mold by itself is somewhat problematic, and that together they are hugely problematic.

    Get rid of the Lyme and the problem gets a lot better but doesn’t go away entirely. That’s what we see from the LLMD’s. But it would seem that even a little LDS can exacerbate the effect, considering just _how_ under control Lyme has to be in order to promote wellness.

    This would explain why people can benefit from Lyme treatment even when they don’t test positive for Lyme on normal (non-IGeneX) Western blot tests. If they have even a LITTLE bit of active Lyme, it may provide enough LDS to cause the effect. Only if you get rid of ALL of it would the effect subside, according to this hypothesis.

    If you can get mold down to zero, maybe you don’t need to worry about Lyme. I'm getting the feeling that LPS by itself doesn't have all that much of an effect.


    The other thing that this researcher states exacerbates the effects of the mold is H2O2.

    What do you suppose he’s suggesting here? What would cause there to be a lot of hydrogen peroxide in to be having an effect on us (and exacerbating the effects of the mold) at a particular point in time?

    If I knew that, maybe I could make an effort to eliminate the H2O2 component.


    I’m so glad that you’re taking a look at this. I’ve been puzzling over it for a while now. Thinking about it in the context of what the virus might do is interesting and (it seems to me) potentially really important.


    Lisa

    [This Message was Edited on 10/12/2009]
  6. RunningAntelope

    RunningAntelope New Member

    Lisa, read Cheney's most recent blog about "XMRV and its intracellular toxicity based on redox shifts."

    He essentially reaffirms and vindicates what I said above about the retrovirus being capable of activating NF-kappa B and shifting the redox state to encourage further replication and exacerbate the virulence of co-infecting viruses by knocking out the enzymes I discussed above, in addtion to gluthathione peroxidase (the one I neglected to mention, but an important one). From what I remember studying about retroviruses in virology, I think he stands on solid ground. The key will be research investigating XMRV's complementary gene sequences capable of switching off the transcription of our own DNA responsible for the proteins involved in the production and induction of these enzyme systems.

    He also discussed the potential for activation of NF-kappa-B independently of the retrovirus, which further propagates the virus and could explain the multiple sensitivity issues with vaccinations, heavy metal toxicity (apart from exposure devoid of toxicity), and the "impressive reactions to mold." He gives two citations, so you may want to go read the research articles for yourself.

    If he's correct, the problem with giving glutathione or promoting intracellular glutathione in such a shifted redox state with all of its hyper-ROS problems, is that you could merely be upregulating the oxidation of gluthathione and undermining "free energy."

    nofool, thanks for the constructive contribution. You may want to consider refining your stew of brain drugs.


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  7. RunningAntelope

    RunningAntelope New Member

    His further comments about increased UV sensitivity of "TAT-gene infected cells," promoting the potential for increased skin cancers like basal cell and squamous cell carcinomas was very intriguing to me, since I have developed both of these, and the basal cell hit me when I was 28 years young and at the peak of my disease state! TAT is one of the very complementary gene sequences (complement of our own DNA that codes for these enzyme systems mentioned above capable of rendering them inactive) possessed by one HIV virus. I can only assume that XMRV has similar genomic sequences.

    Also the comments in regard to glutathione peroxidase dependence on selenium and viral myocarditis in mice with selenium deficiency was also interesting because of the point mutation in the most easily oxidized region of the viral DNA. These bugs have unbelievable mutation proliferation rates and survivability strategies.
  8. Slayadragon

    Slayadragon New Member

    Well, that's interesting. Has he talked about that before?

    I'm thinking that Judy Mikovits must have told him about that after her visit to Erik.

    I'm going to dig into those articles today.

    Lisa
  9. RunningAntelope

    RunningAntelope New Member

    Yes, he's intimated it via the xenobiotic stress and superoxide production reacting with toxins (mycotoxins or otherwise) that we discussed on the cohorts thread I believe in the context of P450. And he talks to Dr. Shoemaker frequently, so it's always been on his radar screen, though I think he vigorously disagrees with Shoemaker's approach. I've never SEEN him emphasize mold in particular though. Still, it's in keeping with his theory and the P450/NADPH decoupling. One VERY IMPORTANT point he makes is the potentially deleterious impact of drugs on P450.
    [This Message was Edited on 10/12/2009]
  10. Slayadragon

    Slayadragon New Member

    >One VERY IMPORTANT point he makes is the potentially deleterious impact of drugs on P450.

    What kind of drugs?