Current Research Projects

Our project explores the possibility that antibodies (Ab)s with proteolytic activity may exist in some patients with ME-CFS. These unique autoantibodies, also called Abzymes (Abz)s, combine the natural structure and function of an antibody with properties of an enzyme. Abzs may target DNA, RNA, protein, peptides or other macromolecular structures, where they bind to the substrate with high specificity and break it down in situ. For abzymes that target proteins, because of their very low catalytic powers compared to general proteases like trypsin, tissue damage may occur over months before symptoms manifest. Our recently published results featured in Biochemistry1 (Fig. 1) suggest the presence of myelin-basic protein (MBP)-specific Abzs in plasma of some ME-CFS patients (Fig. 2). In support of ME-CFS patient MRI data that indicate white matter disease, Abzs may digest MBP in the central and peripheral nervous systems and cause demyelination. Therefore, in patients with neurological symptoms (e.g., nerve pain and muscle weakness), Abzs might be useful in the clinical setting as a metric to correlate with disease severity. Moving forward with this project, we plan to isolate and characterize Abzs from both cerebral spinal fluid and plasma of ME-CFS patients to determine the following: 1) level of substrate digestion as a marker of disease severity, 2) degree of binding to citrullinated MBP, 3) exact Abz cut sites of MBP, 4) Abz specificity to non-MBP substrates including DNA, collagen, insulin and gamma interferon, and 5) Abz drug inhibition by glatiramer acetate and other small molecules.

References:

1. Jensen, M. A., Dafoe, M. L., Wilhelmy, J., Cervantes, L., Okumu, A. N., Kipp, L., Nemat-Gorgani, M., and Davis, R. W. (2024) Catalytic Antibodies May Contribute to Demyelination in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, Biochemistry 63, 9-18; PMC10765373

Pathological fatigue is often associated with disruptions in processes critical to energy production. These may include defects in mitochondrial function, such as impaired substrate uptake, transport, and metabolism, reduced availability of enzyme cofactors, or the presence of enzyme inhibitors affecting various ATP production pathways.

To study these complexities, the Biolog MitoPlate™, originally optimized by Dr. Laurel Crosby, offers a robust, cell-based phenotyping assay designed to interrogate and characterize mitochondrial function through colorimetric analysis. The MitoPlate™, developed by Biolog Inc., consists of a 96-well plate, with each well containing a unique dried substrate.

Using freshly isolated peripheral blood mononuclear cells (PBMCs) and a permeabilization agent, cellular mitochondria are made accessible for analysis. When cells are plated onto the MitoPlate™, a colorimetric reaction based on tetrazolium salt reduction is triggered. This reaction is captured by the OmniLog® incubator-camera system, providing insights into the metabolic activity of the cells and their ability to process specific substrates. The intensity of the colorimetric change reflects the electron flow to the terminal electron acceptor in the electron transport chain, where a redox dye is reduced, producing a purple color.

The MitoPlate™ includes a diverse range of substrates, such as cytoplasmic and mitochondrial substrates, L-Malic-dependent substrates, and those involved in the tricarboxylic acid (TCA) cycle. Recent findings have demonstrated a reduced utilization of specific substrates in participants with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) compared to healthy control samples. This reduction may indicate impaired generation of NADH and FADH, suggesting mitochondrial dysfunction as a contributing factor in ME/CFS pathology.

Using high multiplex PCR developed at Stanford Genome Technology Center, we study viral reactivation in ME/CFS, Long-COVID, trauma and sepsis. The viruses we focused on are EBV, HHV6 and CMV. Currently, we have ~450 PCR amplicons to sequence each and every gene in HHV6 and EBV genome.

We use blood plasma samples collected from ME/CFS, COVID, trauma and sepsis patient to perform PCR and Illumina sequencing. We found the viral reactivations are common for patients in an infection stage and disappeared once recovered.

This high multiplex PCR technology is also used to study gene variants in BH4 pathway for ME/CFS patients and comparing to healthy controls.

Comparative studies on the biomechanics of red blood cells (RBCs) from ME/CFS patients and healthy controls involve collaboration with a group in Chemical Engineering (Dr. Jiandi Wang, PI) at UC Davis, with the goal of developing a diagnostic device. The project is based on the premise that deoxygenation weakens plasma membrane-cytoskeleton interactions, resulting in membrane detachment, and a linear increase in RBC capillary velocity with decreased local oxygen tension (PO2). Such PO2-regulated behavior has been shown to be different in RBCs from ME/CFS patients and healthy controls, suggesting that it may serve as a potential biomarker. Moreover, a number of drugs which have been previously reported to bring about changes in cell rigidity appear to be useful in affecting RBC mechanical properties toward a more normal behavior. These studies have been conducted using microfluidic devices involving images recorded by a high-speed camera. More recently, we have been working on the design and fabrication of an impedance-based platform for patient diagnosis. The device will replace the current platform which requires the use of a high-speed camera, extensive image processing and analysis. We hope it will be accurate, inexpensive and easy to operate in different clinical settings. 

This data analysis project is led by Khoa Nguyen, in collaboration with Julie Wilhemy in Ronald Davis’s laboratory at the Stanford Genome Technology Center, Stanford University. 

The project aims to explore the role of innate immunity, a key cellular regulator of host defense against infection, in ME/CFS pathophysiology. High-throughput single-cell proteomic analysis (CYTOF) of the Stanford ME/CFS cohort (n=13-14) revealed an accumulation of monocytes in the blood samples of patients compared to healthy controls (HC). Furthermore, ME/CFS monocytes produced more TNF-a upon in vitro stimulation, suggesting that prior infectious exposure might have pre-programmed them towards an inflammatory phenotype. In congruence with this hypothesis, we observed elevated expression of CD57 (a putative marker of CMV exposure) in NK cells from ME/CFS patients compared to HC. Additionally, ME/CFS NK cells were highly activated, evidenced by increased expression of IFN-g and Granzyme B compared to HC counterparts. Together, these findings suggest that abnormally activated monocytes and NK cells might be involved in inflammation-related pathologies in ME/CFS. 

Further ongoing high-dimensional characterization of other immune-cell repertoires in the Stanford ME/CFS cohort will provide novel insights about abnormalities in cellular communication networks that underlies ME/CFS pathophysiology. Another future research direction of this project is to independently validate the findings above by secondary analysis of publicly available “omics” studies of ME/CFS, which will have important implications for diagnostic/therapeutic discovery.

Recent advances in genomics, gene editing, and synthetic biology have fueled a strong demand for higher quality, longer oligonucleotide production. However, in the past ten years, no major advances have been made in the field of solid-phase phosphoramidite chemistry, and strand length continues to be limited by its diminishing yields. Currently it costs up to $0.30 per bp to assemble a single gene ($300 / kb). This high cost is largely due to the workload of eliminating errors in assembled gene products and then choosing and sequence-validating the correct clone. Here, errors are the source of failure strands, which are invariably generated during solid-phase synthesis. Traditionally, columns and micro-wells are packaged with solid support, containing thousands of beads or glass shards, where DNA is immobilized on the surface or within channels of the support. As such, deletions are the result of 1) spent reagents and unwanted byproducts that become trapped within the support and carry over into consecutive cycles, and 2) when reagents do not completely contact all DNA molecules where beads overlap one another due to stacking. To circumvent these limitations, we propose a novel method that allows us to control the actions of an individual bead through dielectrophoresis on a plasmonic surface (Figure 1). Here, reactions are tuned to completely encapsulate each bead with minimal volume reagent droplets for high-precision synthesis. Because each bead is isolated in solution, byproducts cannot become trapped, and each has maximum contact with all synthesis reagents; it is this intimate 1:1 ratio of bead to reagent that will significantly increase the base addition efficiency allowing production of ultra-long strands of DNA > 1000 bases (). Moreover, this method will drop the cost of gene production > 100-fold (< 0.3 cents per bp) and transform research in all major fields of biomedicine.

The nanoneedle biosensor is a cost-effective, versatile, ultrasensitive and high-throughput nanoelectronics device. We adapted this array to detect the electrical impedance of ME/CFS peripheral blood mononuclear cells (PBMCs). After analysis of over 20 ME/CFS patients and 20 HCs, we have established that ME/CFS PBMCs exhibit a characteristic impedance pattern when subjected to salt stress that is significantly different than controls. This work has been published in the Proceedings of the National Academy of Sciences (USA).

Further studies are underway to understand the differences between the patients’ PBMCs and those of healthy controls. To date, we know that a patient’s serum can cause HC’s PBMCs to adopt a ME/CFS impedance pattern. Preliminary fractionation experiments indicate that the factor responsible for this effect may reside in the exosome fraction of plasma. The nanoneedle assay can also be used to screen potential compounds that can revert ME/CFS impedance patterns to that of HCs. 

Future efforts are being directed toward understanding the biological causes of the ME/CFS impedance pattern in the nanoneedle assay. One direction of research is to identify the specific cell population in PBMCs that may be generating the impedance pattern. Another approach is to identify the specific component(s) in patient sera that can cause HC cells to produce ME/CFS impedance patterns. In order to understand the specificity of the nanoneedle assay for ME/CFS, PBMCs from MS patients, a disease that is somewhat similar to ME/CFS, will be tested.  This work has been sponsored by the Open Medical Foundation and the NIH.

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Overview

Developed by Rahim Esfandyarpour, PhD, in collaboration with Ronald W. Davis, PhD, at Stanford University.

The nanoneedle biosensor is a cost-effective, versatile, ultrasensitive and high-throughput nanoelectronics device. This device measures the electrical impedance of cells from a single drop of blood.

This test is able to distinguish ME/CFS patients from healthy controls.

An analysis of over 40 ME/CFS patients and controls has established that cells from the blood of ME/CFS patients exhibit a characteristic impedance pattern when subjected to salt stress that is significantly different than the controls.

Future tests of this device will be made to:

·       Determine if differences are observed between ME/CFS patients and other related diseases.

·       Whether impedance signals are indicative of illness severity by testing a series of patients at altered severity.

·       To test candidate drugs to see if the salt stress impedance signal can be returned to healthy control levels, revealing the potential of the nanosensor array platform for use in drug screening.

The technology will be optimized for easy clinical adoption and scaled up so that numerous FDA-approved drugs can be simultaneously screened as potential treatments

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 More  Developing blood-based diagnostic and drug screening technology

There is currently no biological test to diagnose ME/CFS and as a result, diagnosing patients is a lengthy and costly process, constituting a fundamental impediment in patient care. This lag in diagnosis also erects barriers to research, complicating patient recruitment and potentially engaging a heterogeneous sample of patients with only superficially similar conditions.

Dr. Davis’s team is dedicated to developing inexpensive tests that can be easily used in a doctor’s office.  Patients will be measured on multiple diagnostic platforms, enabling  comparisons of efficacy to determine what combination of platforms would be most useful  for diagnostic testing.

The Collaborative Center will continue this work to engineer a blood-based diagnostic device that would also be useful  for in vitro drug screening. Dr. Davis’ team has already tested chemicals in two  platforms, some of which have made the patient samples behave more like healthy samples. To validate these findings and test large numbers of samples and candidate drugs, they will further develop and optimize the technology. Eventually, the developed technology will be shared across the ME/CFS research community to accelerate its evaluation and adoption as a clinical diagnostic assay. The Stanford Genome Technology Center has developed a number of very successful diagnostic assays for other purposes that have been commercially exported and are now in clinical use. Dr. Davis’ team has experience in the complex process of developing and implementing  assays that have been approved for clinical use.

Four technologies are being developed that could provide a diagnostic tool  for ME/CFS:

Nanoneedle assay

Red Blood cell deformability assay

Mitochondrial function test

Magnetic levitation platform

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In February 2019, we published in Clinical Hemorheology and Microcirculation our results that demonstrated a significant decrease in deformability of red blood cells (RBCs) from ME/CFS patients compared to HCs, which may have origins in oxidative stress. We hypothesize that altered microvascular perfusion due at least in part to reduced RBC deformability can be a possible cause for ME/CFS symptoms. Our data also suggest that RBC deformability may serve as a potential biomarker for ME/CFS, albeit further studies are necessary for non-specific classification of the disease.

We have assembled a collaborative team to work on several approaches to studying RBC deformability in ME/CFS patients. In addition to Ron Davis and personnel at the Collaborative Research Center at Stanford, three other PIs and their labs are involved: Anand Ramasubramanian of San Jose State, who was one of the corresponding authors of the RBC deformability publication; Juan Santiago, director of the Stanford Microfluidics Laboratory; and Eric Shaqfeh, a Stanford expert in simulation and analysis of blood flow in microfluidics. Together the group has designed and fabricated 10 design variations for a microfluidic platform that can yield imaging data suitable for comparison to computer simulations generated by the use of  High-Performance Computing Resources by the Shaqfeh group. Microfluidic devices suggested from the simulations will be tested with the ultimate goal being the creation of a sensitive diagnostic device. This project is sponsored by the Open Medical Foundation.

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Overview

Developed by Mohsen Nemat-Gorgani, PhD, of Stanford University, and Anand Ramasubramanian, PhD, of San Jose State University. In collaboration with Juan Santiago, PhD, and Eric Shaqfeh, PhD, and Ronald W. Davis, PhD, at Stanford University.

Several studies have implicated a role of oxidative stress in ME/CFS. Red blood cells (RBCs) are potent scavengers of oxidative stress and their shape changes appreciably in response to oxidative stress; this has been observed in certain inflammatory conditions including obesity and diabetes.

The shape of RBCs determine how well these cells can move through blood vessels so it seems pertinent to determine if RBCs in ME/CFS patients are affected. This has led to the development of a microfluidic device that mimics blood flow through microcapillaries.

Preliminary studies have shown that RBCs from ME/CFS patients had an altered rate of movement through microcapillaries and that RBCs from ME/CFS patients had reduced deformability.

The significant decrease in deformability of RBCs from ME/CFS patients may have origins in oxidative stress and suggests that altered microvascular perfusion can be a possible cause for ME/CFS symptoms.

This work has been accepted for publication in Clinical Hemorheology and Microcirculation and also has been accepted as an abstract for the American Society of Hematology 60th Annual Meeting.

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Developed by Julie Wilhelmy, PhD, in collaboration with Ronald W. Davis, PhD, at Stanford University.

The Seahorse instrument measures oxygen during energy production processes that occur in the mitochondria. A protocol using the Seahorse has revealed a significant difference between activated T-cells of ME/CFS patients and healthy controls.

The instrument is commercially available, which will allow other laboratories to easily reproduce the results.

We have developed several next-generation tools that leverage advances in synthetic biology and CRISPR/Cas9 genome editing. These tools include a core technology called Recombinase Directed Indexing (REDI), which transforms complex oligonucleotide libraries into high-density ordered arrays of yeast clones, each containing a unique sequence-verified oligonucleotide of interest. In addition, Multiplexed Accurate Genome Editing with Short, Trackable, Integrated Cellular Barcodes (MAGESTIC) enables high-throughput modification of the yeast genome at unprecedented scale and precision. We are applying these tools to produce new natural product chemicals by expressing biosynthetic pathways from other organisms in an optimized yeast host. For example, heterologous expression of several plant genes, combined with modifications to native yeast metabolism, is being used to produce the natural product Strictosidine, a common precursor to several medically and commercially valuable chemical entities that is extremely expensive to isolate from natural sources. We are also applying our technologies towards engineering of proteins. Projects include systematically evaluating the effects of synonymous genetic changes on translation efficiency, mapping amino acids substitutions that alter drug binding and resistance, and engineering novel enzymatic activities of commercial value. This work is sponsored by the NIH.

Our immune system consists of a vast repertoire of T cells, with an estimated diversity of over 108 T cell receptor (TCR) beta chains alone, permitting the detection of many different foreign antigens. Upon recognition of an infected cell, a T cell expands clonally, retaining its original TCR sequences, which are so unique they act as a barcode. While TCR repertoires have been investigated in ME/CFS using bulk RNA-Seq, differences in activated T cells (which constitute only 2–5% of this population) would be easily obscured by this approach. We have developed a robust single-cell method for TCR sequencing and phenotyping. This effort is sponsored by the Open Medical Foundation and the NIH.

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Overview

This study is led by Michael Sikora, in collaboration with Mark M. Davis, PhD, Lars M. Steinmetz, PhD, and Ronald W. Davis, PhD, at Stanford University.

Beginning in 2016, the aim of this study was to establish the role of T cells and the immune system in ME/CFS by examining the genetic material in T cells - immune cells that identify and kill infected cells. The rationale is that aspects of of genetic material in these cells can inform whether they are actively fighting an infection and potentially what the cause of the infection is.

Many studies have shown that the immune system is affected in ME/CFS patients, e.g., low activity of NK cells, altered levels of cytokines (signaling molecules of the immune system), and the likelihood of a microbial infection preceding the illness. None of these studies are yet to implicate T cells or define their activity in ME/CFS patients.

The investigation of the immunological basis of ME/CFS will have several parts that overall will help determine if ME/CFS is an autoimmune disease and what immune factors may be triggering ME/CFS or sustaining it as a chronic disease.

Dr. Mark Davis’ team is investigating the clonal expansion of T cells in ME/CFS, including what they might be targeting – viruses, bacteria, or self (autoimmune).

Dr. Ron Davis’ team has invented a highly accurate, cost-effective method for HLA gene sequencing and a very sensitive method for detecting viral DNA as a sign of viral infections, which he is using in this project.

Dr. Lars Steinmetz’ team has developed effective methods for sequencing RNA from single T cells, which they are using to understand how T cell behavior may be different in ME/CFS.

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The hypothesis that environmental stressors interact with a potentially common genetic lesion led to the discovery of common, damaging mutations in the IDO2 gene in the Severely Ill Patient (SIP) cohort. These mutations unmask the well-known substrate inhibition of its partner enzyme, IDO1. This nonlinear dependence of IDO1 on its amino acid substrate, tryptophan (Trp), opens the door to what, in the language of nonlinear systems theory, is called a bistable system, which can be forced to shift by environmental stimuli to a stable pathological steady state. This work is funded by the Open Medical Foundation.

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Overview

This study is led by Robert D. Phair, PhD, of Integrated Bioinformatics, Inc, in collaboration with Julie Wilhelmy, PhD, and Ronald W. Davis, PhD, at Stanford University.

Beginning in 2018, the aim of this study was to test the hypothesis developed by Dr Phair that  a crucial component of metabolism in ME/CFS patients appears to be “trapped” in an unhealthy state.

The metabolic trap theory emerged from the genetic and metabolomics data from the Severely ill Patients Study (SIPS). Using previously published work, Dr Phair has developed a computational program that can model the flow of metabolism throughout the body and its cells.  Using this technology, Dr Phair can determine any points that may disrupt the flow using genomic and metabolic information and this has led to the hypothesis of a metabolic trap that occurs in ME/CFS patients at the point in metabolism where tryptophan is converted to serotonin and kynurenine.

To validate the hypothesis, experiments were initiated to measure the levels of tryptophan and kynurenine in cells from ME/CFS patients. This is a complicated procedure as taking cells out of the body without disrupting their inherent metabolism is fraught with problems.

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Chronic Fatigue Syndrome, or Myalgic Encephalomyelitis (ME/CFS) is a chronic debilitating condition that is characterized by fatigue, post-exertional malaise, unrefreshing sleep, and either cognitive impairment or orthostatic intolerance. The critical barrier to progress in this field is that Chronic Fatigue Syndrome is diagnosed based on subjective symptoms, rather than specific, quantifiable, and actionable biomarkers. The lack of objective biomarkers raises the possibility that Chronic Fatigue Syndrome is a disease with several different etiologies. Although the cause(s) of ME/CFS remain unclear, there are several non-specific indications of organic disease that differentiate patients from healthy controls, including increased levels of oxidative and nitrosative stress, neuroinflammation, immune system abnormalities, and disturbed energy metabolism. Notably, a recent study evaluating thyroid function in ME/CFS patients identified a subset of individuals with “Low Free T3 Syndrome,” which is characterized by a reduced conversion of tetraiodothyronine (thyroxine, T4) to triiodothyronine (T3).

In order to address whether environmental factors contribute to a potential disease mechanism in ME/CFS, we performed a metallomics analysis of hair from 47 patients and 31 healthy controls. We identified a subset of patients with hair mercury levels exceeding the World Health Organization (WHO) reference range of 2.0 ugHg/g hair. Heavy metals, especially methylmercury, exert toxic effects by directly inhibiting various enzymes, but these metals also have a strong affinity for the essential trace metal, selenium. It is possible that mercury exposure reduces the bioavailability of selenium, even when concentrations of selenium in tissues do not appear to be limiting. The significance of this observation is that selenium is a required cofactor for thyroid hormone metabolism and management of antioxidant defenses, which have both been shown to be deficient in patients diagnosed with ME/CFS. Our central hypothesis is that environmental exposure to methylmercury reduces the bioavailability of selenium, and causes a reduction in various selenoenzyme activities in patients with chronic, unexplained fatigue. This project is sponsoered by the Open Medical Foundation.

The severely ill patient (SIP) Big Data study continues to yield intriguing insights into the pathology of myalgic encephalopathy/chronic fatigue syndrome (ME/CFS). Natural killer (NK) cells are an immune cell subtype implicated in this disease, and recent analysis of the SIPs data has found polymorphisms in a complex family of receptors expressed by NK cells that are in association with ME/CFS. This family of receptors is called killer cell immunoglobulin-like receptors (KIRs), and they regulate NK cells by binding to major histocompatibility complex proteins, which are encoded by the human leucocyte antigen (HLA) system and are central to human immunity. To date, five patient-enriched variants in the KIR genes (3DL3, 2DL4 and 3DL2) have been found in association with ME/CFS. However, both the HLA and KIR genes are challenging to sequence due to sequence diversity as well as homologous and repetitive regions. To meet this challenge, Chia-Jung Chang and Wenzhong Xiao have collaborated with Marcelo Fernandez Vina, an expert in clinical HLA sequencing at Stanford, to establish new standards for HLA and KIR sequencing so that the potential role of NK cells can be better understood in ME/CFS by sampling a larger number of ME/CFS patients. This research is funded by the Open Medical Foundation.

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Overview

This study is co-led by Ronald W. Davis, PhD, and Wenzhong Xiao, PhD.

The aim of the Severely Ill Patient Study (SIPS), which started in 2015, was to conduct a comprehensive “Big Data” analysis on severely ill ME/CFS patients in order to begin an exploration to find the molecular basis of ME/CFS. The rationale of the study was that molecular differences would show the strongest signal in severe cases of the illness when compared to healthy controls.

Over 1,000 tests have been conducted per patient, which is the largest (to our knowledge) dataset ever generated in any patient.  

Using samples of saliva, blood, urine and feces, this big data study examined the following:

• Genome

• Gene expression on individual immune cell types

• Metabolomics

• Microbiome

• Cell-free DNA sequencing and quantitation

• Cytokines

• Clinical tests 

The value of a study of this magnitude is that it provides a foundation of invaluable information to generate hypotheses and to develop diagnostics and treatments. Further, all samples from one patient are taken at one point in time, enabling all measurements to reflect exactly what was happening in the body at that one time point. This allows the possibility for the discovery of biomarkers involving the relationships between different body systems that would not ordinarily be found.

This large dataset has been made available online to all interested researchers in the ME/CFS Data Center at Stanford, with the purpose of inspiring research globally, eliminating duplication of effort, and building a collaborative network of scientists.

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This study is led by Fereshteh K. Jahaniani, PhD, in collaboration with Michael P. Snyder, PhD, and Ronald W. Davis, PhD, at Stanford University.

Beginning in 2016, the aim of this study was to extend the Severely ill Patient Study (SIPS) and conduct a comprehensive “Big Data” analysis on ME/CFS patients and their families.  The rationale of this study is that by using the unaffected family members as a control, the differences observed in the ME/CFS patients will be more specific to the disease and less related to differences in genetics, environment, and diet.

Almost as extensive as the SIPS study, blood from each patient is being used to examine the following:

·       Genome

·       Gene expression

·       Metabolomics

·       Proteomics

·       Cytokines

By comparing patients to healthy blood relatives, we are more likely to understand what genes cause or contribute to the development of ME/CFS. This data will also be integrated with the SIPS data.

The team anticipates that this study will help define meaningful subgroups of patients, biomarkers that could be useful in diagnosis and monitoring of disease progression, and molecular defects that could be targeted with new treatments.