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Friday, September 30, 2016

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

( - 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 at: .

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



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.


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


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.


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%.


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.



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, #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.



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