Proton magnetic resonance spectroscopy of basal ganglia in chronic fatigue syndrome.

Fatigue is a common symptom of neurological diseases that affect basal ganglia function. We used proton magnetic resonance spectroscopy ((1)H MRS) to study the metabolic functions of the basal ganglia in chronic fatigue syndrome (CFS) to test the hypothesis that fatigue in CFS may have a neurogenic component. (1)H MRS of left basal ganglia was carried out in eight non-psychiatric patients with CFS and their results were compared to age- and sex-matched healthy asymptomatic healthy controls. A highly significant increase in the spectra from choline-containing compounds was seen in the CFS patient group (p < 0.001). In the absence of regional structural or inflammatory pathology, increased choline resonance in CFS may be an indicator of higher cell membrane turnover due to gliosis or altered intramembrane signalling.

Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection.

Gene expression of key enzymes in 2 antiviral pathways (ribonuclease latent [RNase L] and RNA-regulated protein kinase [PKR]) was compared in 22 patients with chronic fatigue syndrome (CFS), 10 patients with acute gastroenteritis, and 21 healthy volunteers. Pathway activation in the group of patients with infections differed significantly from that of the other 2 groups, in whom there was no evidence of upregulation. Therefore, assay of activation is unlikely to provide the basis for a diagnostic test for CFS.

Cytokines and the acute phase response in post-treatment reactive encephalopathy of Trypanosoma brucei brucei infected mice.

Stimulation of the acute phase response during infection of mice with Trypanosoma brucei brucei (T. b. brucei) was investigated in an experimental model of the post-treatment reactive encephalopathy (PTRE), a common side-effect of anti-trypanosome therapy. Plasma levels of the acute phase proteins (APP), haptoglobin (Hp) and serum amyloid P (SAP) increased by day 7 post-infection, but by day 20 had fallen to an intermediate level. This was accompanied by induction of the cytokines, interleukin (IL)-6 and tumour necrosis factor-alpha (TNFalpha) in both liver and brain. Treatment of mice on day 21 with a subcurative dose of diminazene aceturate (Berenil), a procedure known to induce a mild PTRE, cleared the parasite from the circulation with plasma APP and liver expression of mRNA for IL-6 and TNFalpha returning to the levels in the controls. Cytokine mRNA for both IL-6 and TNFalpha was detected in the brains of animals with developing PTRE although TNFalpha was not significantly greater than in the control group. A further subcurative dose of Berenil, leading to a more severe PTRE, was associated with elevated serum concentrations of Hp and SAP, increased TNFalpha mRNA in the liver and detectable IL-6 and TNFalpha mRNA in the brain. mRNA for IL-1alpha was expressed in brain and liver samples from all animals. A severe PTRE caused a systemic acute phase response which was not apparent with a mild PTRE. The pattern of cytokine mRNA induction was similar following both drug treatments. However, the difference in APP production could be caused by a breakdown in the blood-brain barrier during severe PTRE allowing cytokine synthesised in the brain to enter the circulation and maintain a systemic response.

Latent Varicella-zoster virus in human dorsal root ganglia.

To understand further the molecular events underlying the process of Varicella-zoster virus (VZV) latency in human ganglionic tissues, in situ hybridisation (ISH) for VZV RNA and DNA, and PCR in situ amplification for VZV DNA were used in human dorsal root ganglia from 12 individuals (3 normal and 9 who had died with AIDS). The results showed that (a) two separate regions of the VZV genome, represented by genes 4 and 40, were detected in neurons in two normal and three AIDS ganglia, (b) evidence of transcription of VZV genes 4, 21, 29, and 63 was found in normal and AIDS cases, and (c) VZV DNA and RNA for the same gene (gene 29) was detected in neurons in serial tissue sections in three cases. Thus more than one region of the VZV genome is present in neurons during VZV ganglionic latency, and the presence of both a VZV gene and its corresponding RNA transcript can be shown to occur in the same localised region of DRG tissue.

Latent varicella-zoster virus is located predominantly in neurons in human trigeminal ganglia.

Varicella-zoster virus (VZV) is a human herpesvirus that causes varicella (chicken pox) as a primary infection and, after a variable period of latency in trigeminal and dorsal root ganglia, reactivates to cause herpes zoster (shingles). Both of these conditions may be followed by a variety of neurological complications, especially in immunocompromised individuals such as those with human immunodeficiency virus (HIV) infection. There have been a number of conflicting reports regarding the cellular location of latent VZV within human ganglia. To address this controversy we examined fixed wax-embedded trigeminal ganglia from 30 individuals obtained at autopsy, including 11 with HIV infection, 2 neonates, and 17 immunocompetent individuals, for the presence of latent VZV. Polymerase chain reaction (PCR), in situ hybridization, and PCR in situ amplification techniques with oligonucleotide probes and primer sequences to VZV genes 18, 21, 29, and 63 were used. VZV DNA in ganglia was detected in 15 individuals by using PCR alone, and in 12 individuals (6 normal non-HIV and 6 positive HIV individuals, but not neonatal ganglia) by using PCR in situ amplification. When in situ hybridization alone was used, 5 HIV-positive individuals and only 1 non-HIV individual showed VZV nucleic acid signals in ganglia. In all of the VZV-positive ganglia examined, VZV nucleic acid was detected in neuronal nuclei. Only occasional nonneuronal cells contained VZV DNA. We conclude from these studies that the neuron is the predominant site of latent VZV in human trigeminal ganglia.

Genomic and template RNA transcription in a model of persistent enteroviral infection.

Enteroviruses have been implicated in persistent infections of the nervous system and in certain paralytic motor neuron syndromes. Enteroviral persistence may depend on defective transcription, resulting in the abnormal production of equal amounts of genomic and template RNA strands rather than the normal ratio of 60-100:1. An in vitro model of a persistent coxsackie virus in human skeletal muscle cells was investigated using in situ hybridisation and a semiquantitative parallel, complementary, reverse transcriptase polymerase chain reaction. The ratio of genomic to template RNA was found to be approximately 60:1. We conclude that enteroviral persistence in this in vitro model is not dependent on altered transcription. In vivo, other viral and host factors should be considered.

Search for picornaviruses at onset of inflammatory myopathy.

Picornaviruses may not play a role as persistent agents in the inflammatory myopathies, but it is still thought likely that they may act as triggers of an autoimmune process. Forty one muscle biopsy specimens, taken from three weeks to six months (mean four months) after onset, were examined using three different picornaviral primers and PCR. Moderate to severe disease activity was evident in all specimens. The results were compared with those of 18 biopsy specimens examined later in the disease course, and with specimens from 27 patients with non-inflammatory myopathies. All results were negative. Thus, even as early as three weeks after clinical disease appears, picornaviruses are not detectable in these disorders.

Endogenous cyclo-oxygenase substrates mediate the neuroactivity of evening primrose oil in rats.

The role of cyclo-oxygenase and or its substrate(s) on the neuroactivity of evening primrose oil was investigated on the basis that a blockade of cyclo-oxygenase activity using aspirin would inhibit neuroactivity of primrose oil if this effect was mediated by prostanoids. Streptozotocin diabetic rats and controls were all given large doses of aspirin, but only subgroups of them received primrose oil. Saphenous sensory A- and C-fibre, and sciatic motor conduction velocities were measured to assess neuroactivity of primrose oil. Aspirin enhanced the neuroactivity of primrose oil thus indicating that prostanoids are unlikely to mediate this neuroactivity, but also suggesting that substrates of cyclo-oxygenase are neuroactive. It is likely that cyclo-oxygenase antagonises neuroactivity of primrose oil by competing with the process for substrates. Thickly myelinated sensory A-fibres were most affected by primrose oil suggesting that the predominant sensory symptoms in diabetic neuropathy could be due to the sensitivity of sensory nerves to the metabolic aberration in diabetes. Normal nerve function is probably preserved by cyclo-oxygenase during an influx of neuroactive fatty acids from the gut, since inhibition of the enzyme rendered non-diabetic nerves vulnerable to dietary primrose oil.

Studies on enterovirus in patients with chronic fatigue syndrome.

A large study on 121 patients with the chronic fatigue syndrome (CFS) that examined muscle biopsy samples for enterovirus by means of polymerase chain reaction analysis was carried out. The results were compared with those obtained from 101 muscle biopsy specimens from patients with a variety of other neuromuscular disorders (OND), including neurogenic atrophies, dystrophies, and mitochondrial, metabolic, and endocrine myopathies. Thirty-two (26.4%) of the biopsy specimens from the group of patients with CFS were positive, compared with 20 (19.8%) from the group of patients with OND, a difference that was not significant. This finding is in contrast to those of our previous smaller study in which significantly more patients with CFS than control subjects (53% [32 of 60] vs. 15% [6 of 41]) had enterovirus RNA sequences in their muscle. It was concluded that it is unlikely that persistent enterovirus infection plays a pathogenetic role in CFS, although an effect in initiating the disease process cannot be excluded.

Enteroviruses and postviral fatigue syndrome.

Postviral fatigue syndrome (PFS) occurs both in epidemics and sporadically. Many of the original epidemics were related to poliomyelitis outbreaks which either preceded or followed them. The core clinical symptoms are always the same: severe fatigue made worse by exercise, myalgia, night sweats, atypical depression and excessive sleep. The other common symptoms include dysequilibrium disorders and irritable bowel syndrome. We have detected enteroviral genome sequences in muscle biopsies from cases of PFS, using specific enteroviral oligonucleotide primers in the polymerase chain reaction (PCR). In addition, whole virus particles can be demonstrated in PCR-positive muscle, using solid-phase immuno-electron microscopy. An increase in the number and size of muscle mitochondria was found in 70% of PFS cases, suggesting an abnormality in metabolic function. Evidence of hypothalamic dysfunction was present, particularly involving 5-hydroxytryptamine metabolism. A putative model of PFS, based on persistent enteroviral infection in laboratory mice, revealed resolving inflammatory lesions in muscle with, however, a marked increase in the production of certain cytokines in the brain. This model may help to explain the pathogenesis of PFS.

Search for retrovirus in the chronic fatigue syndrome.

AIM: To examine peripheral blood and skeletal muscle from patients with chronic fatigue syndrome for exogenous retrovirus. METHODS: Blood samples from 30 patients and muscle biopsy specimens of 15 patients were examined for retroviral sequences by DNA extraction, polymerase chain reaction (PCR), and Southern blotting hybridisation. Sera were examined for human foamy virus by western immunoblotting and indirect immunofluorescence techniques. RESULTS: No differences between the patient and control populations was found for any of the PCR primer sets used (gag, pol, env, and tax regions of HTLV I/II). An endogenous gag band was observed in both the patient and control groups. All sera were negative for antibody to human foamy virus. CONCLUSION: The results indicate that there is no evidence of retroviral involvement in the chronic fatigue syndrome.

Immunopathology of experimental African sleeping sickness: detection of cytokine mRNA in the brains of Trypanosoma brucei brucei-infected mice.

The immunopathology of Trypanosoma brucei brucei in the central nervous system was studied by using an experimental model of chronic meningoencephalitis in outbred CD-1 mice. Mice infected with T. b. brucei were treated with a subcurative dose of the trypanocidal compound diaminazine aceturate. These mice relapsed and were again drug treated. The brains were examined histologically and by immunocytochemistry to identify activated astrocytes. The polymerase chain reaction was used to detect cytokine RNA transcripts. The infected and treated animals developed severe chronic meningoencephalitis characterized by large numbers of inflammatory cells and widespread astrocyte proliferation. In uninfected controls, only interleukin 1 and beta-actin RNA transcripts were detected while transcripts for beta-actin, tumor necrosis factor alpha, macrophage inflammatory protein 1 and interleukins 1 and 4 were demonstrated in the brains of infected animals. Gamma interferon and interleukin 6 were also detected in a few of the infected animals, but interleukin 2 was not found in any of these animals.

Amplification and identification of enteroviral sequences in the postviral fatigue syndrome.

Evidence from several sources has long suggested that enteroviruses might play a role in the postviral fatigue syndrome (PVFS). We used the most sensitive molecular virological method available at present, the polymerase chain reaction (PCR) amplification technique, to look for enteroviral copies in peripheral blood leucocytes and muscle from a well-defined group of patients. We demonstrated that our PCR method amplified a sequence common to a wide range of enteroviral serotypes. A highly significant number of the muscle biopsies (53%: P = less than 0.001) from the patients were positive for enteroviral sequences. With regard to the leucocyte samples, 16% in both patient and control were positive. The PCR results on the peripheral blood leucocytes were in keeping with serological findings, in showing that the level of exposure to enteroviruses seemed to be the same in patients and controls: it was therefore of the greatest interest that patients were 6.7 times more likely to have enteroviral genome in their muscle. We conclude that persistent enteroviral infection plays a role in the pathogenesis of PVFS, also providing preliminary evidence that severe mitochondrial injury is one of the mechanisms involved.

Enteroviral RNA sequences detected by polymerase chain reaction in muscle of patients with postviral fatigue syndrome.

OBJECTIVE: To determine the presence of enteroviral sequences in muscle of patients with the postviral fatigue syndrome. DESIGN: Detection of sequences with the polymerase chain reaction in a well defined group of patients with the syndrome and controls over the same period. SETTING: Institute of Neurological Sciences, Glasgow. SUBJECTS: 60 consecutive patients admitted to the institute with the postviral fatigue syndrome who had undergone extensive investigation to exclude other conditions. 41 controls from the same catchment area without evidence of fatigue, all undergoing routine surgery. MAIN OUTCOME MEASURES: Routine investigations, serological screen for antibodies to a range of viruses, and presence of enteroviral RNA sequences in muscle biopsy specimens. RESULTS: 15 (25%) patients and 10 (24.4%) controls had important serological findings. 12 patients had neutralising antibody titres of greater than or equal to 256 to coxsackieviruses B1-5 (six positive for enteroviral RNA sequences, six negative); three were positive for Epstein-Barr virus specific IgM (two positive, one negative). Six controls had similar neutralising antibody titres to coxsackieviruses (all negative); one was positive for Epstein-Barr virus specific IgM (negative); and three had titres of complement fixing antibody greater than or equal to 256 to cytomegalovirus (all negative). Overall, significantly more patients than controls had enteroviral RNA sequences in muscle (32/60, 53% v 6/41, 15%; odds ratio 6.7, 95% confidence interval 2.4 to 18.2). This was not correlated with duration of disease, patient and age, or to raised titres of antibodies to coxsackieviruses B1-5. CONCLUSIONS: Persistent enteroviral infection of muscle may occur in some patients with postviral fatigue syndrome and may have an aetiological role.

A negative regulatory sequence near the mouse beta-maj globin gene associated with a region of potential Z-DNA.

The possible regulatory role of DNA sequences situated 5' to the beta-maj globin gene was investigated by two types of assay. First, a long term transformation assay was used to measure the efficiency of transformation of TK- mouse (LATK-) and hamster (BHKTK-) fibroblast cells with DNA molecules made by covalently linking mouse and human DNA fragments to the herpes simplex virus (HSV-1) thymidine kinase (tk) gene in the plasmid pTK1. When the promoter regions from the mouse beta-maj globin or the human epsilon-globin genes are substituted for the viral promoter in the tk gene transformation occurs with 10-20% of the efficiency of the original plasmid. A fragment (H1), containing sequences between 344 and 1413 bp upstream from the mouse beta-maj globin cap site, almost completely abolishes transformation when inserted next to hybrid tk genes containing the mouse beta-globin or human epsilon-globin promoter but has no effect on the intact tk gene. The effect can be demonstrated with the H1 fragment in either orientation relative to the tk gene. Secondly, in transient expression assays the H1 fragment strongly inhibits transcription when covalently linked to the tk gene under control of either globin promoter, but not when linked to the tk gene with its own promoter. The H1 fragment contains 53 bp of purine-pyrimidine alternation (ACAT)n as part of a larger region potentially capable of adopting a Z-DNA conformation.

In vivo assembly of regularly spaced nucleosomes on mouse beta maj-globin DNA cloned in an SV40 recombinant.

Short-term transformation of HeLa cells with an SV40 recombinant carrying a 7.0-Kb mouse genomic globin DNA was studied. It was found that 48 h after transfection the donor DNA was present in the cell nucleus at high copy episomal numbers with the globin gene in regularly spaced nucleosomal form and transcribed into 9S poly A+ RNA. These mini-chromosomes can be isolated in sufficient quantity to allow further biochemical and electron microscopic studies.