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Friday, September 30, 2016

CDMRP-Funded Study Finds 3 Blood Biomarkers of Gulf War Illness, Advances Toward Treatments

(91outcomes.com) - A Minnesota researcher has identified three "readily measurable" biomarkers in the blood of 1991 Gulf War veterans suffering from Gulf War Illness that may help enhance the current case definition for the disease and aid in treatment trials.  

Supported by a U.S. Department of Defense (DoD) research grant, Dr. Ronald Bach of the Minneapolis VA Medical Center and his research team identified the biomarkers, which include specific changes in lymphocytes, monocytes, and C reactive protein.  Additionally, "Lymphocyte, monocyte, neutrophil, and platelet counts were higher in GWI subjects. Six serum proteins associated with inflammation were significantly different in GWI subjects."


Together, these findings add new support to the hypothesis that chronic inflammation is present in veterans with Gulf War Illness and contributes to their debilitating symptoms.  

According to the research publication in the PLos One journal, "Blood Biomarkers of Chronic Inflammation in Gulf War Illness," this discovery will, "significantly augment the symptom-based case definition of GWI. These new observations are highly relevant to the diagnosis of GWI, and to therapeutic trials."


The study was funded by the Gulf War Illness Congressionally Directed Medical Research Program (CDMRP), which in turn is funded by Congress under the auspices of the DoD health program at the request of Gulf War veteran advocates.


The findings have already led to a follow-on treatment trial now underway and also funded by the Gulf War Illness CDMRP.  In the new treatment trial, ill Gulf War veterans can participate to help advance the science and help determine the effectiveness of one of a myriad of potential treatments currently under investigation by CDMRP-funded Gulf War Illness research projects across the U.S.


According to Bach's research team, "A clinical trial that will further evaluate inflammatory parameters and the efficacy of anti-inflammatory therapy in GWI is in progress at our institution.... The results of this clinical trial will provide valuable data to further evaluate the utility of measuring inflammatory biomarkers in the diagnosis of GWI, but validation by studies of other cohorts of veterans with GWI is required."


The Minneapolis-based Gulf War Illness Inflammation Reduction Trial is currently recruiting Gulf War veteran participants.  More information about the new study, including contact information for how to participate, is available from ClinicalTrials.gov at: https://clinicaltrials.gov/ct2/show/NCT02506192 .


The biomarker study was funded by the GWI CDMRP with FY2008 Congressional appropriations ("Biomarkers of Gulf War Veterans’ Illnesses: Tissue Factor, Chronic Coagulopathy, and Inflammation,", GW080080).  The treatment tries was funded by the GWI CDMRP with FY2013 Congressional appropriations ("Gulf War Illness Inflammation Reduction Trial," GW130025).  

###

*******

SOURCE:  PubMed, published in PLoS One, June 28, 2016, Dr. Ronald Bach, principal investigator.


***

ARCHIVED ABSTRACT (full journal article at link above):

. 2016; 11(6): e0157855. 
Published online 2016 Jun 28. doi:  10.1371/journal.pone.0157855
PMCID: PMC4924830

Blood Biomarkers of Chronic Inflammation in Gulf War Illness

Michelle L. Block, Editor

Abstract

Background

More than twenty years following the end of the 1990–1991 Gulf War it is estimated that approximately 300,000 veterans of this conflict suffer from an unexplained chronic, multi-system disorder known as Gulf War Illness (GWI). The etiology of GWI may be exposure to chemical toxins, but it remains only partially defined, and its case definition is based only on symptoms. Objective criteria for the diagnosis of GWI are urgently needed for diagnosis and therapeutic research.

Objective

This study was designed to determine if blood biomarkers could provide objective criteria to assist diagnosis of GWI.

Design

A surveillance study of 85 Gulf War Veteran volunteers identified from the Department of Veterans Affairs Minnesota Gulf War registry was performed. All subjects were deployed to the Gulf War. Fifty seven subjects had GWI defined by CDC criteria, and 28 did not have symptomatic criteria for a diagnosis of GWI. Statistical analyses were performed on peripheral blood counts and assays of 61 plasma proteins using the Mann-Whitney rank sum test to compare biomarker distributions and stepwise logistic regression to formulate a diagnostic model.

Results

Lymphocyte, monocyte, neutrophil, and platelet counts were higher in GWI subjects. Six serum proteins associated with inflammation were significantly different in GWI subjects. A diagnostic model of three biomarkers—lymphocytes, monocytes, and C reactive protein—had a predicted probability of 90% (CI 76–90%) for diagnosing GWI when the probability of having GWI was above 70%.

Significance

The results of the current study indicate that inflammation is a component of the pathobiology of GWI. Analysis of the data resulted in a model utilizing three readily measurable biomarkers that appears to significantly augment the symptom-based case definition of GWI. These new observations are highly relevant to the diagnosis of GWI, and to therapeutic trials.

....

Discussion

Long after the end of the 1990–1991 Gulf War many veterans of this conflict are ill with an unexplained chronic, multi-system disorder recognized by the Department of Veterans Affairs as GWI []. This disorder is characterized by an incomplete understanding of its etiology and pathophysiology, and a case definition based only on symptoms []. The absence of objective diagnostic criteria is a substantial barrier to clinical diagnosis and research. Despite research by multiple investigators, readily measurable parameters that would permit an objective diagnosis of GWI have not previously been identified. The results of the current study provide evidence of alterations in a number of blood parameters that are readily measurable in routine clinical laboratories. We found white blood cell counts and blood biomarkers related to inflammation could discriminate groups that did or did not meet the current symptom-based criteria for case definition of GWI. Our observations are consistent with conclusions expressed in recent literature reviews that immune dysregulation/neuroinflammation are components of the pathobiology of GWI []. Appropriate assays for the presence of chronic inflammation could provide objective evidence that would facilitate the diagnosis of GWI+.
Alterations of leucocyte counts, particularly the neutrophil to lymphocyte ratio, have recently been reported to have prognostic significance in a wide range of diseases []. An elevated neutrophil to lymphocyte ratio, often found to correlate with CRP or IL6 levels [] has been interpreted as evidence of an inflammatory component of the disorder studied. In contrast to other inflammatory conditions, we did not observe an increase in the neutrophil to lymphocyte ratio because the lymphocyte count was elevated rather than decreased as described in other studies.
Support for an inflammatory component of GWI is provided by the significantly higher levels of plasma CRP detected in GWI+ veterans. CRP is an acute phase plasma protein synthesized in the liver which rises rapidly in response to infection or tissue injury []. CRP is frequently employed as a biomarker of IL-6-mediated inflammation, and it may also augment inflammation. CRP exists in two distinct protein confirmations. Native pentameric CRP is the circulating precursor of monomeric CRP which is strongly proinflammatory [].
In some inflammatory disorders, CRP is highly elevated, but in other disorders modest elevations of CRP have been found to be indicators of chronic inflammation with prognostic significance []. In coronary artery disease CRP concentrations found in the general population (1–3 μg/ml) predict increased cardiovascular mortality [].
Leptin, an adipokine produced primarily by white fat tissue, is another biomarker linked to inflammation, and found in the current study to be elevated in GWI+ veterans. Leptin production is elevated in experimental inflammation and in human autoimmune diseases []. Leptin is known to cross the blood-brain barrier and to interact with cells in the hypothalamus, arcuate nucleus, and endothelium, and with leucocytes. These interactions have been shown to result in prolonged neuroinflammation with behavioral changes in experimental animals []. A leptin antagonist mutant demonstrated benefit in experimental autoimmune inflammatory bowel disease []. Leptin also affects hematopoiesis []. It may have contributed to the elevated lymphocyte counts observed in GWI+ subjects, and thus explain why leptin was not an independent correlate of GWI+. Leptin has also been demonstrated to interact with CRP. In vitro studies found leptin to stimulate the expression of CRP by human hepatocytes. In addition, CRP bound to leptin and directly inhibited its binding to its receptors [].
BDNF, another plasma protein elevated in GWI+ subjects, may also be a biomarker of inflammation in GWI. BDNF is a neurotrophin which functions as a major regulator of synaptic plasticity and neurogenesis in the central nervous system []. Blood BDNF derives primarily from the brain [], and has been observed in several diseases accompanied by inflammation—rapid cycling bipolar disorder [], Alzheimer’s disease [], and fibromyalgia []. Multiple animal studies indicate that overexpression of BDNF following nerve injury or peripheral inflammation stimulates synaptic changes that contribute to chronic pain []. Thus, inflammation-induced BDNF could be a mediator of cognitive impairment and chronic pain in GWI. Neither serum nor plasma BDNF correlate with body weight [] or obesity [].
Matrix metalloproteinases (MMPs) are endopeptidases that participate in tissue extracellular matrix degradation and remodeling. MMP activation has been observed in inflammatory and neurodegenerative disorders []. Elevation of plasma MMP-2, MMP-9 or both have been observed in coronary heart disease [], polypoidal choroidal vasculopathy [] and Japanese encephalitis []. We observed higher median MMP-9, but lower median MMP-2 in GWI+ subjects than in GWI- subjects. The reason for the opposing direction of change is unexplained, but precedent exists for differential patterns of MMP-2 and MMP-9 gene expression in coronary heart disease [], chronic obstructive pulmonary disease [], and thoracic aortic aneurysm [].
H-FABP is a fatty acid binding protein expressed primarily in the heart. Release of H-FABP into the blood occurs during cardiac ischemia, strenuous exercise, and neurodegenerative disorders, but low levels have been reported to occur in patients with Down syndrome []. Decreased H-FABP has been postulated to protect against atherosclerosis. In the current study median plasma H-FABP was significantly lower in GWI+ subjects than in GWI- subjects. Possibly relevant to the lower blood levels in GWI+ subjects is the observation that fatty acid binding protein mRNA was substantially decreased in hamster skeletal muscle and heart muscle by LPS-induced inflammation []. It is possible that blood H-FABP is suppressed by inflammation in GWI, and that the suppression is modulated by elevated blood leptin [].
Although inflammatory mediators are implicated, the precise stimuli for the elevations of blood cell counts, CRP, leptin and the alterations in other blood proteins could not be determined by the current study, and the relationship of these parameters to the symptoms experienced by the subjects with GWI can only be hypothesized. However, observations of others noted above suggest that some of the patient’s symptoms may be caused by or accentuated by CRP, leptin, BDNF or other inflammation-related blood proteins.
This study has strengths and limitations that must be considered in assessing its significance. A strength of the study is the classification of subjects deployed to the Gulf War based on accepted case-definition criteria. Another strength of the study is the evaluation of blood parameters that are readily available in clinical laboratories. The limitations of the study include small sample size, restricted geographic, ethnic, and sex composition of the study subjects, assay of blood parameters only once, some plasma protein assays, including cytokines, considered inevaluable due to a high percentage of assays below the level of detection, overlap of biomarker distributions within the normal range, absence of correction for multiple comparisons, a limited number of blood proteins found to be positively related to GWI+ status, and the absence of a confirmation cohort study.
Despite these limitations, the diagnostic model yielded a high positive predictive value for the 50% of the study participants who had an estimated probability of GWI of 70%, although it is recognized that this estimate may be optimistic because the diagnostic model was fit to these particular data. Although the positive association of 6/61 plasma proteins with GWI+ subjects may have occurred by chance, the close functional and biochemical relationships of these proteins suggest that the differences were not random events. Also, the observation that the subsequent addition of other inflammation-related proteins did not improve the predictive probability of the model above that provided by CRP is consistent with a functional relationship of these parameters.
Although not a deficiency of the study, the issue of obesity is a confounding variable for interpretation of the results of the current study as well as other studies of GWI. The median body weights and BMIs of GWI+ and GWI- subjects were not significantly different, and BMI did not enter into the multivariable diagnostic model. Both groups included obese subjects. Similar obesity observations were found in studies of American [] and Australian Gulf War Veterans []. These studies found no differences in BMI of subjects with GWI and those in comparison groups of veterans who were either non-deployed or deployed to areas other than the Persian Gulf. Coughlin, et al [] found no associations between BMI and unexplained multisystem illness in multivariate analysis. Kelsall, et al [] found a relationship between laboratory parameters of inflammation and multisystem illness, but BMI was not related to multisystem illness []. Dursa, et al [] reported average BMIs of 29.8 for Gulf War Veterans and 29.7 for Gulf War era Veterans 20 years after the Gulf War. These figures are quite similar to those we observed in GWI+ subjects (BMI 31) and GWI-subjects (BMI 28). Therefore, elevation of some of the inflammatory parameters observed in the current study may be attributable to obesity, but obesity does not explain the differences between the GWI+ and GWI- groups, and does not diminish the predictive value of the diagnostic model.
In summary, the results of the current study support the hypothesis that chronic inflammation is a component of the pathophysiology of GWI. Multivariable logistic regression analysis resulted in a model with a high positive predictive value for GWI in subjects with symptoms considered to be significant by current case definition criteria. This diagnostic model requires validation in other samples of Gulf War Veterans. A clinical trial that will further evaluate inflammatory parameters and the efficacy of anti-inflammatory therapy in GWI is in progress at our institution (Gulf War Illness Inflammation Reduction Trial, ClinicalTrials.gov #NCT02506192). The results of this clinical trial will provide valuable data to further evaluate the utility of measuring inflammatory biomarkers in the diagnosis of GWI, but validation by studies of other cohorts of veterans with GWI is required.

***

READ THE FULL JOURNAL ARTICLE HERE:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924830/

###

CDMRP-Funded Study Finds 3 Blood Biomarkers of Gulf War Illness, Advances Toward Treatments

(91outcomes.com) - A Minnesota researcher has identified three "readily measurable" biomarkers in the blood of 1991 Gulf War veterans suffering from Gulf War Illness that may help enhance the current case definition for the disease and aid in treatment trials.  

Supported by a U.S. Department of Defense (DoD) research grant, Dr. Ronald Bach of the Minneapolis VA Medical Center and his research team identified the biomarkers, which include specific changes in lymphocytes, monocytes, and C reactive protein.  Additionally, "Lymphocyte, monocyte, neutrophil, and platelet counts were higher in GWI subjects. Six serum proteins associated with inflammation were significantly different in GWI subjects."


Together, these findings add new support to the hypothesis that chronic inflammation is present in veterans with Gulf War Illness and contributes to their debilitating symptoms.  

According to the research publication in the PLos One journal, "Blood Biomarkers of Chronic Inflammation in Gulf War Illness," this discovery will, "significantly augment the symptom-based case definition of GWI. These new observations are highly relevant to the diagnosis of GWI, and to therapeutic trials."


The study was funded by the Gulf War Illness Congressionally Directed Medical Research Program (CDMRP), which in turn is funded by Congress under the auspices of the DoD health program at the request of Gulf War veteran advocates.


The findings have already led to a follow-on treatment trial now underway and also funded by the Gulf War Illness CDMRP.  In the new treatment trial, ill Gulf War veterans can participate to help advance the science and help determine the effectiveness of one of a myriad of potential treatments currently under investigation by CDMRP-funded Gulf War Illness research projects across the U.S.


According to Bach's research team, "A clinical trial that will further evaluate inflammatory parameters and the efficacy of anti-inflammatory therapy in GWI is in progress at our institution.... The results of this clinical trial will provide valuable data to further evaluate the utility of measuring inflammatory biomarkers in the diagnosis of GWI, but validation by studies of other cohorts of veterans with GWI is required."


The Minneapolis-based Gulf War Illness Inflammation Reduction Trial is currently recruiting Gulf War veteran participants.  More information about the new study, including contact information for how to participate, is available from ClinicalTrials.gov at: https://clinicaltrials.gov/ct2/show/NCT02506192 .


The biomarker study was funded by the GWI CDMRP with FY2008 Congressional appropriations ("Biomarkers of Gulf War Veterans’ Illnesses: Tissue Factor, Chronic Coagulopathy, and Inflammation,", GW080080).  The treatment tries was funded by the GWI CDMRP with FY2013 Congressional appropriations ("Gulf War Illness Inflammation Reduction Trial," GW130025).  

###

*******

SOURCE:  PubMed, published in PLoS One, June 28, 2016, Dr. Ronald Bach, principal investigator.


***

ARCHIVED ABSTRACT (full journal article at link above):

. 2016; 11(6): e0157855. 
Published online 2016 Jun 28. doi:  10.1371/journal.pone.0157855
PMCID: PMC4924830

Blood Biomarkers of Chronic Inflammation in Gulf War Illness

Michelle L. Block, Editor

Abstract

Background

More than twenty years following the end of the 1990–1991 Gulf War it is estimated that approximately 300,000 veterans of this conflict suffer from an unexplained chronic, multi-system disorder known as Gulf War Illness (GWI). The etiology of GWI may be exposure to chemical toxins, but it remains only partially defined, and its case definition is based only on symptoms. Objective criteria for the diagnosis of GWI are urgently needed for diagnosis and therapeutic research.

Objective

This study was designed to determine if blood biomarkers could provide objective criteria to assist diagnosis of GWI.

Design

A surveillance study of 85 Gulf War Veteran volunteers identified from the Department of Veterans Affairs Minnesota Gulf War registry was performed. All subjects were deployed to the Gulf War. Fifty seven subjects had GWI defined by CDC criteria, and 28 did not have symptomatic criteria for a diagnosis of GWI. Statistical analyses were performed on peripheral blood counts and assays of 61 plasma proteins using the Mann-Whitney rank sum test to compare biomarker distributions and stepwise logistic regression to formulate a diagnostic model.

Results

Lymphocyte, monocyte, neutrophil, and platelet counts were higher in GWI subjects. Six serum proteins associated with inflammation were significantly different in GWI subjects. A diagnostic model of three biomarkers—lymphocytes, monocytes, and C reactive protein—had a predicted probability of 90% (CI 76–90%) for diagnosing GWI when the probability of having GWI was above 70%.

Significance

The results of the current study indicate that inflammation is a component of the pathobiology of GWI. Analysis of the data resulted in a model utilizing three readily measurable biomarkers that appears to significantly augment the symptom-based case definition of GWI. These new observations are highly relevant to the diagnosis of GWI, and to therapeutic trials.

....

Discussion

Long after the end of the 1990–1991 Gulf War many veterans of this conflict are ill with an unexplained chronic, multi-system disorder recognized by the Department of Veterans Affairs as GWI []. This disorder is characterized by an incomplete understanding of its etiology and pathophysiology, and a case definition based only on symptoms []. The absence of objective diagnostic criteria is a substantial barrier to clinical diagnosis and research. Despite research by multiple investigators, readily measurable parameters that would permit an objective diagnosis of GWI have not previously been identified. The results of the current study provide evidence of alterations in a number of blood parameters that are readily measurable in routine clinical laboratories. We found white blood cell counts and blood biomarkers related to inflammation could discriminate groups that did or did not meet the current symptom-based criteria for case definition of GWI. Our observations are consistent with conclusions expressed in recent literature reviews that immune dysregulation/neuroinflammation are components of the pathobiology of GWI []. Appropriate assays for the presence of chronic inflammation could provide objective evidence that would facilitate the diagnosis of GWI+.
Alterations of leucocyte counts, particularly the neutrophil to lymphocyte ratio, have recently been reported to have prognostic significance in a wide range of diseases []. An elevated neutrophil to lymphocyte ratio, often found to correlate with CRP or IL6 levels [] has been interpreted as evidence of an inflammatory component of the disorder studied. In contrast to other inflammatory conditions, we did not observe an increase in the neutrophil to lymphocyte ratio because the lymphocyte count was elevated rather than decreased as described in other studies.
Support for an inflammatory component of GWI is provided by the significantly higher levels of plasma CRP detected in GWI+ veterans. CRP is an acute phase plasma protein synthesized in the liver which rises rapidly in response to infection or tissue injury []. CRP is frequently employed as a biomarker of IL-6-mediated inflammation, and it may also augment inflammation. CRP exists in two distinct protein confirmations. Native pentameric CRP is the circulating precursor of monomeric CRP which is strongly proinflammatory [].
In some inflammatory disorders, CRP is highly elevated, but in other disorders modest elevations of CRP have been found to be indicators of chronic inflammation with prognostic significance []. In coronary artery disease CRP concentrations found in the general population (1–3 μg/ml) predict increased cardiovascular mortality [].
Leptin, an adipokine produced primarily by white fat tissue, is another biomarker linked to inflammation, and found in the current study to be elevated in GWI+ veterans. Leptin production is elevated in experimental inflammation and in human autoimmune diseases []. Leptin is known to cross the blood-brain barrier and to interact with cells in the hypothalamus, arcuate nucleus, and endothelium, and with leucocytes. These interactions have been shown to result in prolonged neuroinflammation with behavioral changes in experimental animals []. A leptin antagonist mutant demonstrated benefit in experimental autoimmune inflammatory bowel disease []. Leptin also affects hematopoiesis []. It may have contributed to the elevated lymphocyte counts observed in GWI+ subjects, and thus explain why leptin was not an independent correlate of GWI+. Leptin has also been demonstrated to interact with CRP. In vitro studies found leptin to stimulate the expression of CRP by human hepatocytes. In addition, CRP bound to leptin and directly inhibited its binding to its receptors [].
BDNF, another plasma protein elevated in GWI+ subjects, may also be a biomarker of inflammation in GWI. BDNF is a neurotrophin which functions as a major regulator of synaptic plasticity and neurogenesis in the central nervous system []. Blood BDNF derives primarily from the brain [], and has been observed in several diseases accompanied by inflammation—rapid cycling bipolar disorder [], Alzheimer’s disease [], and fibromyalgia []. Multiple animal studies indicate that overexpression of BDNF following nerve injury or peripheral inflammation stimulates synaptic changes that contribute to chronic pain []. Thus, inflammation-induced BDNF could be a mediator of cognitive impairment and chronic pain in GWI. Neither serum nor plasma BDNF correlate with body weight [] or obesity [].
Matrix metalloproteinases (MMPs) are endopeptidases that participate in tissue extracellular matrix degradation and remodeling. MMP activation has been observed in inflammatory and neurodegenerative disorders []. Elevation of plasma MMP-2, MMP-9 or both have been observed in coronary heart disease [], polypoidal choroidal vasculopathy [] and Japanese encephalitis []. We observed higher median MMP-9, but lower median MMP-2 in GWI+ subjects than in GWI- subjects. The reason for the opposing direction of change is unexplained, but precedent exists for differential patterns of MMP-2 and MMP-9 gene expression in coronary heart disease [], chronic obstructive pulmonary disease [], and thoracic aortic aneurysm [].
H-FABP is a fatty acid binding protein expressed primarily in the heart. Release of H-FABP into the blood occurs during cardiac ischemia, strenuous exercise, and neurodegenerative disorders, but low levels have been reported to occur in patients with Down syndrome []. Decreased H-FABP has been postulated to protect against atherosclerosis. In the current study median plasma H-FABP was significantly lower in GWI+ subjects than in GWI- subjects. Possibly relevant to the lower blood levels in GWI+ subjects is the observation that fatty acid binding protein mRNA was substantially decreased in hamster skeletal muscle and heart muscle by LPS-induced inflammation []. It is possible that blood H-FABP is suppressed by inflammation in GWI, and that the suppression is modulated by elevated blood leptin [].
Although inflammatory mediators are implicated, the precise stimuli for the elevations of blood cell counts, CRP, leptin and the alterations in other blood proteins could not be determined by the current study, and the relationship of these parameters to the symptoms experienced by the subjects with GWI can only be hypothesized. However, observations of others noted above suggest that some of the patient’s symptoms may be caused by or accentuated by CRP, leptin, BDNF or other inflammation-related blood proteins.
This study has strengths and limitations that must be considered in assessing its significance. A strength of the study is the classification of subjects deployed to the Gulf War based on accepted case-definition criteria. Another strength of the study is the evaluation of blood parameters that are readily available in clinical laboratories. The limitations of the study include small sample size, restricted geographic, ethnic, and sex composition of the study subjects, assay of blood parameters only once, some plasma protein assays, including cytokines, considered inevaluable due to a high percentage of assays below the level of detection, overlap of biomarker distributions within the normal range, absence of correction for multiple comparisons, a limited number of blood proteins found to be positively related to GWI+ status, and the absence of a confirmation cohort study.
Despite these limitations, the diagnostic model yielded a high positive predictive value for the 50% of the study participants who had an estimated probability of GWI of 70%, although it is recognized that this estimate may be optimistic because the diagnostic model was fit to these particular data. Although the positive association of 6/61 plasma proteins with GWI+ subjects may have occurred by chance, the close functional and biochemical relationships of these proteins suggest that the differences were not random events. Also, the observation that the subsequent addition of other inflammation-related proteins did not improve the predictive probability of the model above that provided by CRP is consistent with a functional relationship of these parameters.
Although not a deficiency of the study, the issue of obesity is a confounding variable for interpretation of the results of the current study as well as other studies of GWI. The median body weights and BMIs of GWI+ and GWI- subjects were not significantly different, and BMI did not enter into the multivariable diagnostic model. Both groups included obese subjects. Similar obesity observations were found in studies of American [] and Australian Gulf War Veterans []. These studies found no differences in BMI of subjects with GWI and those in comparison groups of veterans who were either non-deployed or deployed to areas other than the Persian Gulf. Coughlin, et al [] found no associations between BMI and unexplained multisystem illness in multivariate analysis. Kelsall, et al [] found a relationship between laboratory parameters of inflammation and multisystem illness, but BMI was not related to multisystem illness []. Dursa, et al [] reported average BMIs of 29.8 for Gulf War Veterans and 29.7 for Gulf War era Veterans 20 years after the Gulf War. These figures are quite similar to those we observed in GWI+ subjects (BMI 31) and GWI-subjects (BMI 28). Therefore, elevation of some of the inflammatory parameters observed in the current study may be attributable to obesity, but obesity does not explain the differences between the GWI+ and GWI- groups, and does not diminish the predictive value of the diagnostic model.
In summary, the results of the current study support the hypothesis that chronic inflammation is a component of the pathophysiology of GWI. Multivariable logistic regression analysis resulted in a model with a high positive predictive value for GWI in subjects with symptoms considered to be significant by current case definition criteria. This diagnostic model requires validation in other samples of Gulf War Veterans. A clinical trial that will further evaluate inflammatory parameters and the efficacy of anti-inflammatory therapy in GWI is in progress at our institution (Gulf War Illness Inflammation Reduction Trial, ClinicalTrials.gov #NCT02506192). The results of this clinical trial will provide valuable data to further evaluate the utility of measuring inflammatory biomarkers in the diagnosis of GWI, but validation by studies of other cohorts of veterans with GWI is required.

***

READ THE FULL JOURNAL ARTICLE HERE:  https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4924830/
###

Wednesday, September 28, 2016

VA Study Confirms Worse Sleep Quality, Higher Sleep Apnea Risk in Gulf War Illness


(91outcomes.com) - A new study by the U.S. Department of Veterans Affairs (VA) shows that veterans suffering from Gulf War Illness (GWI) have greater risk for obstructive sleep apnea than similar veterans without the debilitating condition.

The study, led by longstanding VA GWI researcher Dr. Linda Chao, also found that veterans with GWI had significantly greater severity of insomnia and worse sleep quality.

Interestingly, the study found a close correlation between insomnia and severity and GWI status.

The study concluded that its findings provide evidence supporting the need for treating sleep disturbances in GWI patients.

###


**********

ABSTRACT SOURCE:  PubMed, published in Military Medicine, September 18, 2016, Dr. Linda Chao, principal investigator

https://www.ncbi.nlm.nih.gov/pubmed/27612364


ARCHIVED ABSTRACT:
 2016 Sep;181(9):1127-34. doi: 10.7205/MILMED-D-15-00474.

Insomnia Severity, Subjective Sleep Quality, and Risk for Obstructive Sleep Apnea in Veterans With Gulf War Illness.

Abstract

Despite the fact that sleep disturbances are common in veterans with Gulf War Illness (GWI), there has been a paucity of published sleep studies in this veteran population to date. Therefore, the present study examined subjective sleep quality (assessed with the Pittsburgh Sleep Quality Index), insomnia severity (assessed with the Insomnia Severity Index), and risk for obstructive sleep apnea (assessed with the STOP questionnaire) in 98 Gulf War veterans. Veterans with GWI, defined either by the Kansas or Centers for Disease Control and Prevention criteria, had greater risk for obstructive sleep apnea (i.e., higher STOP scores) than veterans without GWI. This difference persisted even after accounting for potentially confounding demographic (e.g., age, gender) and clinical variables. Veterans with GWI, defined by either the Kansas or Centers for Disease Control and Prevention criteria, also had significantly greater insomnia severity and poorer sleep quality than veterans without GWI (p < 0.05), even after accounting for potentially confounding variables. Furthermore, there were significant, positive correlations between insomnia severity, subjective sleep quality, and GWI symptom severity (p ≤ 0.01). In stepwise linear regression models, insomnia severity significantly predicted GWI status over and above demographic and clinical variables. Together these findings provide good rationale for treating sleep disturbances in the management of GWI. 
Reprint & Copyright © 2016 Association of Military Surgeons of the U.S.
[PubMed - in process]

CDMRP Study Confirms, Builds on Previous Findings that Gulf War Toxins Lead to Long-Term Brain Inflammation

(91outcomes.com) - A study funded by a federal medical research program aimed at developing treatments for Gulf War Illness has found new insight into possible treatment pathways for the debilitating condition that affects as many as one-third of the veterans of the 1991 Gulf War.


The study also confirmed previous findings of persistent neurological inflammation in a mouse model following Gulf War toxic exposures. 

Most notably, according to the study's findings, "the persistent neuroinflammation evident in our model presents a platform with which to identify novel biological pathways, correlating with emergent outcomes that may be amenable to therapeutic targeting. Furthermore, in this work we confirmed our previous findings that GW [Gulf War] agent exposure causes neuropathological changes.... We also extended upon our previous work to cover the lifespan of the laboratory mouse using a battery of neurobehavioral techniques."


The study on which these findings are based was funded by the treatment-focused Gulf War Illness Research Program (GWIRP), within the Congressionally Directed Medical Research Program (CDMRP) administered by the U.S. Department of Defense health program.  The study was funded in 2011 using FY10 CDMRP funds (GW100076).  


For more information, see the project's abstract of what was proposed at: 
http
://cdmrp.army.mil/search.aspx?LOG_NO=GW100076




****

SOURCE:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4709860/  (Full study publication available at this link)


ARCHIVED ABSTRACT:

Front Integr Neurosci. 2015; 9: 71. 
Published online 2016 Jan 12. doi:  10.3389/fnint.2015.00071
PMCID: PMC4709860

A Chronic Longitudinal Characterization of Neurobehavioral and Neuropathological Cognitive Impairment in a Mouse Model of Gulf War Agent Exposure

Abstract

Gulf War Illness (GWI) is a chronic multisymptom illness with a central nervous system component that includes memory impairment as well as neurological and musculoskeletal deficits. Previous studies have shown that in the First Persian Gulf War conflict (1990–1991) exposure to Gulf War (GW) agents, such as pyridostigmine bromide (PB) and permethrin (PER), were key contributors to the etiology of GWI. For this study, we used our previously established mouse model of GW agent exposure (10 days PB+PER) and undertook an extensive lifelong neurobehavioral characterization of the mice from 11 days to 22.5 months post exposure in order to address the persistence and chronicity of effects suffered by the current GWI patient population, 24 years post-exposure. Mice were evaluated using a battery of neurobehavioral testing paradigms, including Open Field Test (OFT), Elevated Plus Maze (EPM), Three Chamber Testing, Radial Arm Water Maze (RAWM), and Barnes Maze (BM) Test. We also carried out neuropathological analyses at 22.5 months post exposure to GW agents after the final behavioral testing. Our results demonstrate that PB+PER exposed mice exhibit neurobehavioral deficits beginning at the 13 months post exposure time point and continuing trends through the 22.5 month post exposure time point. Furthermore, neuropathological changes, including an increase in GFAP staining in the cerebral cortices of exposed mice, were noted 22.5 months post exposure. Thus, the persistent neuroinflammation evident in our model presents a platform with which to identify novel biological pathways, correlating with emergent outcomes that may be amenable to therapeutic targeting. Furthermore, in this work we confirmed our previous findings that GW agent exposure causes neuropathological changes, and have presented novel data which demonstrate increased disinhibition, and lack of social preference in PB+PER exposed mice at 13 months after exposure. We also extended upon our previous work to cover the lifespan of the laboratory mouse using a battery of neurobehavioral techniques.
Keywords: Gulf War, pyridostigmine bromide (PB), permethrin (PER), mouse model, neuropathology, neurobehavioral

***

Discussion

GWI is a chronic multisymptom illness with a CNS component. The pathobiology of GWI remains to be fully elucidated, however, a growing body of evidence suggests that immune and inflammatory dysregulation may be a persistent feature of GWI (Rook and Zumla, ; Skowera et al., ; Peakman et al., ; Broderick et al., ; Smylie et al., ; Craddock et al., ; Johnson et al., ; O'Callaghan et al., ; O'Donovan et al., ). There is also a large body of evidence on the relation between exposure to pesticides and elevated rates of chronic diseases where inflammation is a major component. These include several types of cancers, diabetes, neurodegenerative disorders like Parkinson's disease, Alzheimer's disease, Amyotrophic Lateral Sclerosis (ALS), and recently, GWI (Bonetta, ; Sherer et al., ; Abdollahi et al., ; de Souza et al., ; Mostafalou and Abdollahi, ; Baltazar et al., ). The common feature of chronic disorders is a perturbation in cellular homeostasis, which can be induced via pesticides' primary mechanisms of action. These include disruption of ion channels, enzymes, and receptors (Mostafalou and Abdollahi, ). During the First Persian Gulf War, PB was used as a prophylactic agent against possible exposure to nerve gas agents such as sarin and soman (Sapolsky, ). The protective property of PB is due to its ability to shield the active site of the AChE from attack and subsequent irreversible inhibition by the nerve agents. PER is a broad-spectrum insecticide in the pyrethroid chemical family that works by quickly paralyzing the nervous systems of insects by interfering with the sodium channels. PER was employed as a pesticide by military personnel during the war, where uniforms and nets were pre-soaked with PER (Binns et al., ). Chronic neuroinflammation may be associated with chronic pain, fatigue, and cognitive impairment, and is recognized as one of the main symptom features of GWI in veterans of the Persian Gulf War (Fukuda et al., ; Steele, ; David et al., ; Vythilingam et al., ). GWI patients typically have altered pro- and anti-inflammatory cytokine expression in peripheral immune cells (Skowera et al., ; Broderick et al., ; Khaiboullina et al., ), and it has been suggested that exposure to PB and permethrin (PER) may have altered the balance of cytokine expression in veterans with GWI (Whistler et al., ; Broderick et al., ). PB and PER also modulate ACh-dependent immune mechanisms via inhibition of acetylcholinesterase (AChE) activity, through competitive and non-competitive mechanisms respectively, resulting in elevated peripheral ACh levels (Rao and Rao, ; Peden-Adams et al., ). GW agents have also been implicated in inducing immune responses similar to those seen with Th2 cell activation in the periphery via an ACh mediated mechanism (Punareewattana et al., ; Sullivan and Krieger, ; Nayak et al., ; Dantzer et al., ). Over-stimulation of the brain's immune response can shift it from homeostasis to a pro-inflammatory state which can result in deleterious effects on the central and peripheral nervous systems. These aberrant functions include nonspecific immune damage to neurons and impaired synaptic connections that accompany symptoms of cognitive impairment (Dantzer et al., ; Dilger and Johnson, ). To date, there are no effective treatments for GWI, and thus identification of biological pathways associated with long-term sequelae of exposure to GW agents is vital to understanding the pathogenic mechanisms of GWI and for developing novel therapies for treatment. Therefore, in this animal model of GW exposure, the consequences of combined PB+PER exposure in C57BL6/J mice were examined over a 2 year period, from 11 days to 22.5 months post exposure to GW agents. This work was conducted in order to set up a platform from which to identify biological mechanisms responsible for GW-agent induced pathobiology by spanning the life-time of the animal, so, we would be able to identify specific therapeutic targets in the future.
This study chronicles a longitudinal study in a mouse model of GWI by characterizing the chronic neurobehavioral and neuropathological outcomes following acute early life exposure to GW agents (PB+PER). A battery of neurobehavioral tests were conducted in a single cohort of mice from 11 days to 22.5 months post exposure to GW agents or vehicle control. At 11 days post exposure, no differences were observed in anxiety or any overt signs of locomotor impairment. However, at 13 months post exposure PB+PER exposed mice spent significantly larger proportion of time in the open arms(s) of the EPM, and had an increased number of visits to the open arms as compared to their controls. In addition, PB+PER exposed mice spent significantly larger proportion of time in the center (the junction between the closed and the open arms) of the EPM, as compared to their counterparts. These findings indicate disinhibited behavior in these mice, as a consequence of GW agent exposure, as mice normally preferentially spend more time in dark closed spaces. However, unlike our study, a recent study conducted by Parihar et al. () using a rat model of GWI demonstrated increased anxiety related behavior after exposure to PB, PER DEET and stress at a less chronic time point post-exposure (3 months). In addition, anxiety-like features were observed in a different mouse model of GWI after 28 days of exposure to PB, PER, DEET, and stress, as indicated by an increase in time spent at the periphery of the OFT arena (Abdullah et al., ). Our studies are the first to demonstrate increased disinhibition in PB+PER exposed mice at 13 months after exposure. Disinhibition has been linked with dysfunction of the prefrontal cortex, an area which is crucial for decision making (Hains and Arnsten, ; Gruber et al., ). For example, Vasterling and colleagues have demonstrated a pattern of cognitive disinhibition and commission errors in a cohort of Persian Gulf War veterans in tasks which relate to attention and memory performance (Vasterling et al., ). However, with regards to GW agent exposure paradigms, further studies are warranted in order to definitively confirm the involvement of the prefrontal cortex in mediating anxiety-like behaviors seen in our model.
The Three Chamber Test and the RAWM were also performed at approximately 13 months post exposure. During the Three Chamber testing, PB+PER exposed mice showed a lack of social preference. Fiedler et al. reported that Gulf War Veterans (GWV) had a significantly higher prevalence of psychiatric diagnoses as compared to controls, with deployment as a powerful predictor of current depression and anxiety disorders decades after the end of the GW (Fiedler et al., ). Black et al. reported that GWV had a markedly higher prevalence of current anxiety disorders as compared to non-deployed military personnel (5.9 vs. 2.8%) (Black et al., ). Furthermore, anxiety disorders in GWV were associated with co-occurring psychiatric disorders such as panic disorder, generalized anxiety disorder and PTSD and in GWV as compared to controls were each present at rates nearly twice those expected (Black et al., ). Combat and extreme psychological stressors were less common and less sustained in the Gulf War as compared to other wars (including recent Middle East deployments), and PTSD rates are lower in GWV than in veterans of other wars (Binns et al., ). However, in a recent study of GWV, with and without PTSD, those with the co-morbid expression of PTSD showed increased brain activity in areas spanning the amygdala and the anterior cingulate cortex (Bierer et al., ). Since the anterior cingulate cortex has been associated with functions related to rational cognition, reward anticipation, decision making, impulse control and emotion (Bush et al., ; Williams et al., ), any change within this structure may lead to functional changes, such as behavioral disturbances.
Cognitive deficits using this PB+PER paradigm have been previously demonstrated in this model of GW agent at 5 months post exposure (~8.5 months of age) by Zakirova et al. (), therefore, as we wanted to expand the characterization of this mouse model, neurobehavioral testing in this cohort was conducted at much later time points. In addition, in this cohort we explored different mazes such as the RAWM and the BM, that are primarily designed to measure place learning and memory using environmental visuospatial cues. At 13 months post exposure, post-hoc testing revealed no significant differences between PB+PER exposed and control mice by day during RAWM testing when examining goal arm frequency [visits to the goal arm (#)] and goal arm duration [time spent at the goal arm (s)]. When examining the cumulative distance traveled, differences were detected on day 2 of acquisition trial by post-hoc testing, however, no overall differences were detected between the two groups; likewise, no differences were noted when examining their velocities. In addition, differences between exposed and control mice were detected using RAWM testing when examining working memory errors (#) and reference memory errors (#) made by either the exposed or control mice over the 5 day acquisition testing period. No main effect of exposure was observed when examining both parameters, although, significant differences were noted when examining the interaction between exposure and days post exposure for the number of reference memory errors (#). Additionally, apparent trends were noted in the exposed mice when examining both the number of errors made when examining both working and reference memory. Specifically, it appears as though during the first 3 days of RAWM acquisition testing the exposed mice behave poorly as compared to controls when examining working memory errors, however, by day 4 and 5 of acquisition testing the exposed mice perform better and/or on par with their controls littermates. Interestingly, similar trends are observed when examining the number of reference memory errors. Albeit, purely speculative, we suspect that this acute period of exercise may have improved and/or augmented the working and reference memory performance in PB+PER exposed mice. Exercise has been shown to improve memory acquisition and retrieval in mice (Van der Borght et al., ). In addition, exercise has been shown to significantly increase the number of maturing neurons, indicating that an increase in neurogenesis may be linked to the beneficial effects of exercise (Van der Borght et al., ). Furthermore, an acute period of exercise combined with working memory training has been shown to have synergistic and long-lasting effects on general cognitive performance in mice when voluntary running wheel access was combined with radial arm maze testing (Smith et al., ). Therefore, these data may indicate that acute exercise in the form of RAWM acquisition testing may have beneficial but transient effects on both working and reference memory in PB+PER exposed mice. Overall these data suggest that although learning abilities appear to be intact in PB+PER exposed mice, although some discrete differences in working and reference memory errors were observed when examining the two groups by acquisition trial days, at 13 months post exposure to GW agents.
It is also worth to consider that different maze tasks are able to measure different types of cognitive tasks; therefore, the fact that we used RAWM testing instead of BM testing, which in principal are both designed to measure place learning and memory using environmental visuospatial cues, harbor their own inherent differences. For instance, as Hodges () highlighted, maze tasks differ from one another in many different ways: (a) the types of apparati, which may vary in environmental settings e.g., water vs. land) to multifaceted routes (such as the RAWM); (b) to different types of visuospatial, associative or sensory cues; (c) to various task requirements, which range from random search strategy/exploration to complex and structured sequences of choices; and (d) motivation, such as the opportunity for escape, or find shelter, or to explored novel objects in a new location. Given this multiplicity, it is likely that mazes showcase a variety of neuronal processes that may contribute to spatial learning and memory. Thus, the cognitive skill sets measured using one behavioral test may not be the same as those employed in another, which may create difficulties for the interpretation of exposure-related deficits. In addition, age plays an important role in mice in the context of age sensitivity as it relates to discerning exposure-dependent changes (Kennard and Woodruff-Pak, ). For example, Gower and Lamberty (Gower and Lamberty, ) reported that deficits in acquisition and retention of spatial memory are independent of spontaneous locomotor, sensorimotor, or emotional deficits from middle age (11–12 months) to old age mice (22 months). It has also been demonstrated that genotype has an effect on certain aspects of behavioral tests. For instance, Owen et al. () conducted an extensive comparison of water maze testing performance in 12 mouse strains commonly used for genetic backgrounds and seven hybrid strains. Of those examined, only the C57BL6/J, C57BL10/J, and 129/SvevTac strains were capable of complex learning across multiple tasks, which included water maze testing. The C57BL6/J mice used in these studies were approximately 16.5 months of age and roughly 13 months post exposure to GW agents at the time of RAWM testing. Therefore, the behavioral differences observed in the PB+PER exposed mice during the RAWM acquisition testing, revealing alterations in working memory errors as well as reference memory errors exemplified by poor retention procedural aspects of the behavioral test, as well as poor retention of spatial memory, which are rely on both trial dependent and trial independent storage/processing of memories.
It is important to note that water maze based tests do not only highlight hippocampal-dependent tasks. Woodruff-Pak et al. demonstrated that the process of aging impacts brain structures and associated behaviors differentially, with the cerebellum showing earlier senescence than the hippocampus (Woodruff-Pak et al., ). Therefore, age may affect other brain regions, which may initiate age-related deficits in spatial memory performance. The BM is a hippocampal-dependent behavioral test (Yuede et al., ; Ziehn et al., ). In studies using unexposed, C57Bl6/J mice, older mice (12 and 18 months) made more errors during BM testing than younger mice (3 and 6 months) and relied more on a serial search strategy rather than a spatial strategy (Bach et al., ; Kennard and Woodruff-Pak, ). we have shown deficits in C57BL/6 mice as early as ~8.5 months of age and ~5 months post exposure to GW agents (Zakirova et al., ). In addition, when these GW agent exposed mice were tested at 26 months of age (22.5 months post exposure) using the BM, the exposed mice appeared to make more primary errors as compared to controls, although those differences failed to reach statistical significance. In addition, at this final time point of evaluation, no differences were observed in anxiety-like behaviors in PB+PER exposed mice as compared to controls when examined by EPM. Interestingly, Three Chamber testing at 22.5 months post exposure to GW agents revealed that PB+PER exposed mice exhibited normal sociability and social interaction behaviors, akin to the normal behaviors exhibited by their control counterparts. For instance when examining Sociability and Social interaction, the preference for visiting and spending time with either the empty cage or the novel mouse (stranger 1), PB+PER mice showed a strong preference for spending more time with the stranger 1 mouse over the empty cage, much like the control mice. However, when we examined Social Novelty and Social Memory, the preference for visiting and spending time with now familiar mouse (stranger 1) as compared to the novel mouse (stranger 2), both control mice and PB+PER mice did not exhibit a strong preference for spending time with one mouse over the other. Albeit purely speculative, these data may suggest that certain aspects of social memory/social novelty may be impaired in these mice at such an advanced age. Therefore, we hypothesize that an age effect masked the behavioral differences previously observed in these mice post exposure to GW agents.
The context of age sensitivity, as it relates to discerning exposure-dependent changes, is not only associated with neurobehavioral changes, but also correlates with neuropathological changes. Astrocytes undergo a complex age-dependent remodeling in a brain region-specific manner. The morphological aging of astrocytes was recently investigated in the cortices and the hippocampi of male SV129/C57BL6 mice of different age groups (3, 9, 18, and 24 months) (Rodríguez et al., ). This investigation revealed that GFAP-positive profiles in the hippocampus showed progressive age-dependent hypertrophy, as indicated by an increase in surface, volume, and somata volume at 24 months of age compared with 3-month-old mice (Rodríguez et al., ). On the other hand, aging induced a decrease in GFAP-positive astroglial profiles in the entorhinal cortex (EC) (Rodríguez et al., ). In contrast to these observations, an increase in GFAP immunostaining was observed in PB+PER exposed mice as compared to controls when examining their cerebral cortices 22.5 months post exposure. This increase in astrogliosis in the cerebral cortices of exposed mice is a constant feature of GW exposure in this animal model, which has been demonstrated at 5 months (Zakirova et al., ), 16 months (pers. comm., L. Abdullah), and 22.5 months post exposure (as mentioned here). In addition, there was a slight increase in GFAP staining in the hippocampi of PB+PER exposed mice as compared to controls at 22.5 months post exposure, however, those findings may have been confounded due to the age of the animals (26 months of age—close to the life-span for laboratory mice). Thus, these findings underscore astrogliosis as a persistent and pivotal feature of age-progressive cognitive impairments and neuropathological deficits as a result of GW agent exposure early on in life.
To the best of our knowledge, this work is the first to chronicle such an extensive chronic neurobehavioral characterization using an animal model of GW agent exposure. In addition, neuropathological studies were performed at 22.5 months post exposure to GW agents (well into the end of the life-span for laboratory mice). This lifespan analysis models, in mice, the time that has passed since the current GWI patient population received their pathogenic exposures (nearly two and half decades) as well as the expected progression of the illness, and thus is of considerable relevance for translational research.
In conclusion, the work detailed here describes the successful implementation of this model as a platform in which to identify biological mechanisms responsible for GW-agent induced pathobiology, and thereby to identify therapeutic targets. Validation of one of these targets will be done in the future studies. Specifically, given the persistent signature of chronic but mild neuroinflammation evident in this animal model, future studies will focus on implementing anti-inflammatory agents in order to investigate whether therapeutic intervention earlier in life (middle age) will be beneficial in mediating the effects of inflammation and thereby therapeutically modulating cognitive impairment in this mouse model of GW agent exposure.

Author contributions

Conceived and designed the experiments: GA, FC, and VM. Performed the experiments: ZZ, SH, and LH. Analyzed the data: GC, LA, and ZZ. Contributed reagents/materials/analysis tools: FC and GA. Wrote the paper: ZZ, FC, and GA.

Funding

This research was funded by a Congressionally Directed Medical Research Program award to GA (GW100076), VA merit award to FC and by the Roskamp Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Supplementary material

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fnint.2015.00071

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