Cameron, B., Bharadwaj, M., Burrows, J., Fazou, C., Wakefield, D., Hickie, I., Ffrench, R., Khanna, R. and A. Lloyd. 2006. Prolonged Illness after infectious mononucleosis is associated with altered immunity but not with increased viral load. Journal of Infectious Diseases 193, 664-671.

At least two general possibilities could explain the ongoing debility of the still ill or ‘CFS’ patients in these studies; they could have trouble eliminating the virus, or they could display an response to the virus that persists after the virus has been eliminated. This study examined both possibilities; it examined the viral loads to see if the CFS patients were unable to eliminate the virus, and cytokine levels to see if the CFS patients had a different response to the pathogen. Pro-inflammatory cytokines such as TNF-a, IL-1b and IL-6 are known to cause the symptoms of ‘sickness behavior’ (malaise, fatigue, aching muscles, etc.) so similar to those found in CFS that commonly occur during the early stages (acute phase) of infectious illness

Immune Response - The successful resolution of IM appears to derive from an individual’s ability to mount a ‘broad-based’ cytotoxic T-cell response to EBV. Studies have found that increased levels of EBV specific cytotoxic T-cells are paralleled by declines in the levels of circulating EBV.

Viruses usually enter cells, use the cell’s RNA machinery to produce more viruses and then leave the cell and swarm through the bloodstream looking for more cells to infect. Large numbers of circulating viruses (virions) in the blood stream indicate virus replication is in full swing.

An examination of the cytotoxic T-cell response found that neither people who recovered or who didn’t recover from IM, had a significantly different T-cell response; both groups were able to fairly quickly produce large numbers of EBV specific T-cells that were able to eventually knock down the infection. The researchers also examined how well the immune system responded to the production of two EBV proteins associated with EBV replication. The proteins viruses produce help it to assemble new virions. The T-cells should take note of these new proteins and react by producing a pro-inflammatory cytokine, IFN-7, that helps to regulate the immune response. Both the still ill and the healthy IM patients reacted similarly to the presence of these viral proteins; these patients did not appear to be ill because of their inability to mount an immune response.

Because cytokines can be responsible for many of the symptoms associated with infection, the researchers also examined the two groups to see if the ‘CFS’ patients demonstrated increased or prolonged levels of pro-inflammatory cytokines. The cytokine levels of the ‘CFS’ patients did not, however, differ from those of the recovered patients.

The humoral branch (B-cells) of the immune system has long been thought, because of its delayed appearance, to play a relatively minor role in EBV suppression. Indeed, the antibodies produced by B-cells (IgG anti-EBNA – Epstein-Barr nuclear antigen) appear to signal the beginning of the latency phase when EBV takes up its lifelong residence in B-cells. The presence of anti-EBNA antibodies is believed to be associated with the period of convalescence that follows the acute immune response.

Here, however, a difference was found; the humoral response occurred earlier and was stronger in the CFS-like patients. The researchers posited this meant EBV was able to mount a quicker attack in these patients. They noted that although the both sets of patients mounted an equally powerful cytotoxic T-cell attack that it was ‘somewhat slower’ in the CFS-like ones. They suggested that the earlier humoral response in CFS patients may have been due to enhanced Th2 immune responses. CFS patients have long been thought to display an enhanced Th2 immune response. Cytokines produced during the Th2 immune response activate B-cells, among other things, and increased B-cell activity and antibody production is one of the hallmarks of the Th2 immune response. Interestingly EBV is able to produce a cytokine, IL-10, that induces a Th2 response.

Almost coyly, however, they suggested in the last sentence of the paper, that they did have a idea where the problem lay but aren’t going to share the details of it with us yet. They stated "we propose that alternative neurobiological mechanisms triggered during the severe, acute illness and sustained in the absence of peripheral inflammation underpin prolonged illnesses after EBV infection".

This suggests the problem lies not in the immune system but in the nervous system (probably the brain) and that it is triggered during the initial phase of the illness and then sustained even after the infection has been resolved. It suggests this team has found markers of nervous system dysfunction both early in the illness and later on in the CFS patients.

Several studies have suggested that immune activation may be occurring in the brains of CFS patients. These include the increased white blood cell levels in Natelson’s cerebrospinal fluid study, a distinctive proteome that could reflect glial cell activation in the Baraniuk cerebrospinal fluid study, and the increased choline levels seen in the basal ganglia in several magnetic resonance spectroscopy studies. Albashi believes HHV-6A infection of the CNS contributes to the symptoms seen in CFS (see The Pathogens in CFS, Part I, HHV-6). The Kauschik/Kerr and Vernon gene expression studies have found evidence of both central nervous system and immune dysregulation in CFS. Hopefully the Lloyd team’s next paper explaining their rather cryptic comment will be out soon.

This study found that the gene response in the chronically ill IM (or CFS) patients differed early in the illness, and this difference appeared to reflect the ability of EBV to better evade or exploit these patients immune systems and replicate in the body.

Amazingly all eight of the genes more highly expressed in the CFS-like patients in the early stages of the IM were involved the activation of a phase of the cell  cycle.  The cell cycle is invoked when cells begin to  proliferate. I believe EBV induces B-cells to proliferate in order to create the B-memory cells it hides out in. Four of the genes differentially expressed in the chronically ill group were involved in parts of the immune response EBV is known to evoke or exploit. One, an interferon stimulated gene (ISG20) is evoked by cells infected with viruses. Another, involved in apoptosis, is evoked when the immune system kills off infected cells by invoking their suicide program. Three of the genes were involved in a cellular process called the cell cycle that EBV hijacks in order to replicate in the bodies cells.

An analysis of genes that were upregulated both early and late in the disease process found that over half of them were involved in fatty acid metabolism and mitochondria functioning and several of these genes are known to be associated with increased EBV activity. One of the early proteins produced by EBV (BRLFI), for instance, induces the infected cell to produce an enzyme, called fatty acid synthase (FAS), that produces a fatty acid (palmitate) found in cellular membranes. EBV uses the FAS enzyme and palmitate to awake from latency and begin replication in the cell. Li et. al. noted that the active ingredient in green tea (epigallocatechin gallate) has been found to inhibit FAS activity and EBV replication*. The authors posited that the mitochondrial abnormalities found may have been responsible for the immune alterations seen in  the first study.  Indeed, EBV infected monocytes display reduced production of TNF-a, an important pro-inflammatory cytokine.

The tight fit between the genes found and EBV replication was impressive and suggested increased EBV replication had occurred in the CFS patients. Indeed the authors stated that the gene expression changes seen

"potentially implicates a failure of the host to adequately control viral replication."

Vernon, S, Nicholson, A., Rajeevan, M., Dimulescu, I., Cameron, B., Whistler, T. and A. Lloyd. 2006. Correlation of psycho-neuroendocrine-immune (PNI) gene expression with symptoms of acute infectious mononucleosis. Brain Research 1068, 1-6.

Most gene expression studies have focused on genes found in immune cells in the blood called peripheral blood mononuclear cells (monocytes and lymphocytes). This seems fine for examining immune activity in CFS but what about uncovering information on metabolic or nervous system processes? How in the world can one get information on these processes from immune cells?

Researchers at the CDC recently demonstrated – to their surprise - that almost 2/3rds of 1600 genes known to mediate psychological – neuroendocrine – and immune (PNI) processes were expressed in PBMC’s. This indicates gene expression in the PBMC’s does give a credible snapshot of the biological activity in the body. PBMC gene expression analysis has, in fact, been described as being a kind of molecular biopsy of the body.

This study examined the expression of 1058 genes known to be engaged in PNI processes. First the researchers assessed the symptom presentation of people with infectious mononucleosis. Then they correlated the symptom expression data with gene expression data in order to determine which genes may have contributed to which symptoms.

It is important to note that they did not look at gene expression in CFS patients; this study simply examined what happens to the body to create the symptoms of infectious mononucleosis. Since CFS and infectious mononucleosis have such similar symptoms, however, it is possible that this study uncovered genes involved in generating the symptoms found in CFS. Because this study did not differentiate between chronically fatigued and recovered patients some genes that could be uniquely upregulated in CFS patients may not be presented here. We don’t know if they examined these genes in the second Dubbo study or not. This study, then, does give us a preliminary idea of what might have gone wrong in CFS.

The researchers must have beamed at their results. No genes were significantly associated with fever, malaise or irritability/depression but they found a nice tight fit between gene expression and fatigue, sleep disturbance and neurocognitive problems. This means there is little doubt that these genes play a role in causing these symptoms.

Surprisingly, none of these genes were directly involved in cytokine production. Many researchers believe the symptoms (i.e. sickness behavior) seen in the early stages of an infection are cytokine related. This is an important finding. Skeptics of a physiological interpretation of a post-infective fatigue state have been able to point to the conflicting findings in CFS cytokine studies to assert there is no evidence of an aberrant physiological process. But what if we’re looking in the wrong place? This study strongly suggests that cytokines are not the only agents involved in producing ‘sickness behavior’.

Sleep Problems (Hypocretin/orexin) – How nicely this gene fits this symptom! Low hypocretin levels occur in narcoplepsy, a disease characterized by extreme sleepiness during the daytime and insomnia at night. Narcoleptics get about as much sleep as everyone else but they can’t get it in one shot - they are unable to stay awake or asleep for significant periods of time.

This seems like a strange finding though; increased hypocretin mRNA in EBV patients would lead, one would think, to increased alertness. High hypocretin levels at night, however, could lead to insomnia and that could theoretically lead to increased daytime sleepiness. A disturbance in the hypocretin system by EBV could therefore play a role in the extreme daytime sleepiness found in CFS patients.

So what is MEF2C? MEF2C is a transcription factor, a protein that regulates gene activity; transcription factors enter the nucleus and then either promote or inhibit the transcription of DNA into mRNA. Many CFS patients know of the STAT transcription factor that regulates the transcription of immune genes. One of the genes MEF2C activates promotes the expression of a key viral protein (BZLFI) important in the EBV replication.

This doesn’t appear to tell us much about the fatigue found in infectious mononucleosis patients, though. About half of the IM patients – those without fatigue - did not have high MEF2C levels. Since there is no evidence of increased viral replication in the more fatigued IM patients MEF2C’s role in producing fatigue during IM is something of a mystery.

And what about EBV? Does it play a special role in CFS or not? Is it just one of many pathogens that can trigger CFS or does it have certain features that make more dangerous than others? Is its ability to infect central nervous system cells important or necessary? Are CFS patients at special risk from neurotropic pathogens? Several of the pathogens of interest in CFS (HHV-6, EBV, some mycoplasma’s, coxsackie B) can infect the CNS