Comorbidity of postural orthostatic tachycardia syndrome and chronic fatigue syndrome in an Australian cohort.

OBJECTIVE: Patients with chronic fatigue syndrome (CFS) are frequently diagnosed with comorbid postural orthostatic tachycardia syndrome (POTS), suggesting a shared pathogenesis. The aim of this study was to examine the relationship between demographic characteristics, autonomic functioning and fatigue levels amongst CFS patients with and without comorbid POTS. DESIGN AND SETTING: All patients presenting to the CFS Discovery Clinic between 2009 and 2012 completed a 20-min standing task as part of their initial assessment. Heart rate and pulse pressure were recorded at baseline, at 2-min intervals poststanding, at the end of the task and following a recovery period. Average heart rate and pulse pressure variability were calculated from this data. Age, gender, length of illness and self-reported fatigue scores were also recorded. POTS patients were diagnosed by an orthostatic increase in heart rate >30 beats per min, concomitant symptoms of orthostatic intolerance and no orthostatic hypotension. Differences in autonomic functioning between POTS and CFS patients were compared using independent samples t-tests, whilst logistic and linear regressions were performed to examine the contribution of autonomic functioning to task completion and perceived fatigue, respectively. RESULTS: Comorbidity of CFS and POTS (CFS-POTS) was observed in 11% (33/306) of patients. CFS-POTS patients were significantly younger (P < 0.001), had a shorter length of illness (P = 0.034), experienced greater task difficulty (P = 0.002) and were able to stand for significantly shorter periods compared to the CFS-only patients (P < 0.001). CFS-POTS patients experienced significantly lower baseline diastolic blood pressure (P = 0.002), significantly higher heart rate and lower pulse pressures at each standing measurement. Early heart rate changes (P = 0.002) and overall heart rate change (P < 0.001) were significant predictors of completion status, whereas heart rate variability (P < 0.001) and female gender (P < 0.001) were significant predictors of increased perceived task difficulty. CONCLUSIONS: Haemodynamic and demographic differences between CFS-POTS and CFS-only patients suggest that the former group reflects a distinct subgroup of the CFS population. The findings highlight the utility of screening younger patients with fatigue for POTS, and identified heart rate variability as an important marker of fatigue for CFS patients in general.

Viral co-infection does not reduce the efficacy of vaccination against non-typeable Haemophilus influenzae middle ear infection in a rat model.

The mucosal vaccination of rodents with killed non-typeable Haemophilus influenzae (NTHi) has been previously shown to enhance live NTHi clearance following middle ear challenge. This study assessed the efficacy of mucosal anti-NTHi vaccination during a concomitant viral infection of the respiratory tract. Animals were mucosally immunised with killed NTHi by intra-Peyer's patch primary inoculation and lung (intratracheal) boost. At the time of both immunisations rats were also infected intra-nasally with Sendai virus. Concomitant Sendai virus infection did not influence the efficacy of anti-NTHi vaccination mediated clearance of NTHi from the middle ear. This would suggest that immunisation strategies to prevent bacterial middle ear infection would be effective despite the presence of concomitant viral agents.

Genetic 'budget' of viruses and the cost to the infected host: a theory on the relationship between the genetic capacity of viruses, immune evasion, persistence and disease.

The nature of the pathogen-host relationship is recognized as being a dynamic coevolutionary process where the immune system has required ongoing adaptation and improvement to combat infection. Under survival pressure from sophisticated immune responses, adaptive processes for microbes, including viruses, have manifested as immune evasion strategies. This paper proposes a theory that virus immune evasion can be broadly classified into 'acquisition' or 'erroneous replication' strategies. Acquisition strategies are characteristic of large genome dsDNA viruses, which (i) replicate in the cell nucleus; (ii) have acquired host genes that can be used to directly manipulate responses to infection; (iii) are often latent for the lifetime of the host; and (iv) have little or no serious impact on health. Alternatively, erroneous replication strategies are characteristic of small genome RNA viruses, which are recognized as being the cause of many serious diseases in humans. It is proposed that this propensity for disease is due to the cytoplasmic site of replication and truncated temporal relationship with the host, which has limited or removed the evolutionary opportunity for RNA viruses to have acquired host genes. This has resulted in RNA viruses relying on error-prone replication strategies which, while allowing survival and persistence, are more likely to lead to disease due to the lack of direct viral control over potentially host-deleterious inflammatory and immune responses to infection.

Specific ablation of antiviral gene expression in macrophages by antibody-dependent enhancement of Ross River virus infection.

Ross River virus (RRV) is an indigenous Australian arthropod-borne alphavirus responsible for epidemic polyarthritis (EPA), myalgia, and lethargy in humans. Macrophages and monocytes have been associated with human RRV disease, and previous studies have shown that RRV is capable of infecting macrophages via both a natural virus receptor and by Fc receptor-mediated antibody-dependent enhancement (ADE). Similar to other viruses, such as human immunodeficiency virus and dengue virus, ADE infection results in dramatic RRV growth increases for in vitro macrophage cultures. This study demonstrates that RRV could resist lipopolysaccharide (LPS)-induced antiviral activity in macrophage cultures when infection was via the ADE pathway. Investigation of this infection pathway found that RRV was able to suppress the transcription and translation of key antiviral genes (tumor necrosis factor and inducible nitric oxide synthase) in LPS-stimulated macrophages by disrupting the transcription into mRNA of the genes coding for the associated transcription factors IRF-1 and NF-kappaB. The transcription of non-antiviral control genes was not perturbed by RRV-ADE infection, and de novo protein synthesis also was not significantly affected in RRV-ADE infected cells. The ADE pathway of infection allowed RRV to specifically target antiviral genes in macrophages, resulting in unrestricted virus replication. As ADE has been observed for several virus families and associated with disease and adverse vaccination outcomes, these findings may have broad relevance to viral disease formation and antiviral vaccination strategies.

Studies on the IgA-independent immunological responses in mice to influenza virus challenge after oral vaccination with irradiated whole virus and an erythrocyte complex.

Previous studies have described an oral influenza vaccine comprising whole irradiated virus and an erythrocyte complex (IV-EC), which gave broad-based protection against influenza virus challenge in mice. The present study examined the immune responses generated after live virus challenge of vaccinated mice, particularly to determine whether mice vaccinated with IV-EC had enhanced CTL activity to compensate for the previously reported diminution in lung IgA response. Oral vaccine groups examined were IV-EC, live virus alone (LV) or live virus-erythrocyte complex (LV-EC), compared with irradiated virus and erythrocyte alone controls. The antibody responses of IV-EC and LV-EC vaccinated mice showed significantly elevated lung and serum IgG2a levels post live virus challenge, with no comparable increases in IgG1 levels compared to controls. Spleen cells from IV-EC mice showed an enhanced post-challenge proliferative response to antigen compared with mice that had received live oral vaccines, indicating enhanced cellular activity post IV-EC immunization. However, CTL activity was not enhanced for IV-EC mice, and live virus-vaccinated mice had reduced CTL activity compared with controls, indicating that CTL were not important for post-vaccine protection. Cytokine analysis revealed a predominant IFN-gamma response in spleen cells from orally vaccinated mice, whereas IL-4 was not detected in any lung or spleen culture analysed. The results suggest, therefore, that protection from live influenza challenge after IV-EC or LV-EC vaccination was due to an IFN-mediated IgG2a response. Definitive confirmation of the role of these factors in post-vaccine protection can now be tested in IgG2a-depleted or IFN-gamma gene knockout mouse models.

Macrophage-induced muscle pathology results in morbidity and mortality for Ross River virus-infected mice.

Ross River virus (RRV) is an Australian alphavirus that is often responsible for chronic epidemic polyarthritis and myalgia in humans. Past studies have shown severe disruption of striated muscle fibers to be prominent in RRV pathology in mice; in the present study, macrophages were directly implicated as the primary mediators of muscle damage. General immunosuppressive therapies had only minor effects on mortality and morbidity in RRV-infected mice, with no inhibition of muscle damage. Treatment of mice with macrophage-toxic agents (e.g., silica) prior to RRV infection completely abrogated disease symptoms without significantly affecting titers of virus in organs. Further studies found that clinical signs of infection and muscle damage correlated with a massive influx of macrophages into hind leg muscle, whereas no such infiltrate or damage was observed for silica-treated mice. These observations are significant for the human disease context, as monocytic cells have been detected in the synovial effusions of persons with epidemic polyarthritis.

Erythrocytes enhance the immunogenicity of oral vaccination with gamma irradiated influenza virus: increasing the dose of irradiation results in a significant diminution of lung IgA response.

Previous studies have demonstrated that the ability of gamma-irradiated whole influenza virus to prime for specific anti-influenza antibody responses was dramatically enhanced when delivered in a complex with chicken red blood cell ghosts (cRBC). The purpose of this study was to investigate the effect of increasing the dose of gamma irradiation used to inactivate A/Queensland/6/72 virus on the ability of the virus-cRBC complex to prime for specific influenza responses. Spleen cell proliferation studies confirmed the enhancing effect of the cRBC carrier for oral vaccination with irradiated virus. Cells from mice vaccinated with 30 kGy-irradiated virus only did not respond to influenza stimulation in vitro, whereas cells from mice vaccinated with irradiated virus+cRBC showed significant increases in proliferation to antigen exposure. No significant antibody response or challenge virus clearance was observed in mice orally vaccinated with irradiated (13.1 or 30 kGy) virus alone, even when the dose was increased significantly. Oral vaccination with live virus (+/-cRBC) primed for significant influenza specific IgA responses in the lungs, in addition to IgG responses in the lungs and sera. The dose of irradiation used to inactivate the virus was found to be critical to the profile of antibody response when the virus was delivered in a complex with cRBC. Oral vaccination of Swiss mice with 13.1 or 30 kGy virus (+cRBC) primed for significant serum and lung IgG responses. Lung IgA responses for 13.1 kGy+cRBC vaccinated mice were detected, but 30 kGy+cRBC vaccinated Swiss and CBA/H mice had no significant lung IgA response. The abrogation of IgA response, however, did not lessen the clearance of live challenge virus in outbred mice, suggesting a primary role for IgG and/or CTL response in the control of influenza virus infection post oral vaccination. To ensure direct comparison of virus alone and virus+cRBC treatments, the concentration of virus complexed to the cRBC was determined.

The role for host-immune factors in the in vivo antiviral effects of tumour necrosis factor.

This report examines the host-immune response to vaccinia virus in mice exposed to tumour necrosis factor (TNF). Exposure to TNF was done via two approaches; (1) mice were treated with TNF in complex with a specific anti-TNF antibody (Ab 301) which enhances the antiviral efficacy of this cytokine, prior to vaccinia virus (VV) infection; and (2) mice were infected with a recombinant VV which encodes the murine TNF gene (VV-HA-TNF). The antiviral effect induced by TNF plus Ab 301 in CBA/H mice was not sensitive to sub-lethal gamma irradiation, indicating that a proliferative immune cell population was not responsible for the observed attenuated VV growth. This was further evidenced by NK cell and CTL studies which showed, when compared to controls, that TNF plus Ab 301 treated animals had decreased spleen NK cell and CTL activities. This suggests that a non-specific factor, effective earlier than day 2 postinfection, was responsible for the restriction of VV growth. To further investigate this possibility, mice were treated with either the TNF/Ab 301 regimen, or infected with VV-HA-TNF, and their peritoneal exudate cells (PEC) examined during early VV infection. Control recombinant VV infection resulted in an increase in neutrophil numbers, and TNF/Ab 301 treatment before infection did not increase this further. VV-HA-TNF infection of mice, however, induced a massive but transient increase in the number of neutrophils, suggesting that this cell population was important to the in vivo restriction of VV-HA-TNF growth.

The antiviral activity of tumour necrosis factor on herpes simplex virus type 1: role for a butylated hydroxyanisole sensitive factor.

We have previously shown that specific antibodies (Mab 32/Ab 301) against tumour necrosis factor (TNF) enhance its antiviral activity in vaccinia virus-infected mice. In the present study, TNF alone was found to have antiviral activity against herpes simplex virus-1 (HSV-1). Antibody enhancement was found, both in vivo and in vitro, at lower TNF doses. The magnitude of the TNF-induced antiviral response was dependent upon the genetic background of the mouse. C57BL/6 mice were very sensitive to the antiviral activity of TNF, which was inhibited by the free radical scavenger butylated hydroxyanisole (BHA). TNF plus Mab 32 induced a significant antiviral effect in L929 cells which was associated with pronounced CPE. The CPE was largely reversed in the presence of BHA, and furthermore, TNF antiviral activity was significantly reversed in the presence of BHA. Specific inhibitors of nitric oxide synthetase, lipoxygenase or cyclo-oxygenase did not influence either the CPE or growth kinetics of HSV-1, suggesting that neither reactive nitrogen intermediates nor arachidonic acid metabolites were involved in the antiviral mechanism of TNF. This, together with observed increases in Cu/Zn SOD levels in virus infected cells, suggests that reactive oxygen intermediates may have a role in the direct control of HSV-1 growth and that free radicals may play a part in the antiviral activity induced by TNF.

Was exposure to directly antiviral cytokines during primary infection an important selective pressure in the evolution of unique immune evasion strategies by viruses?

Different virus families are characterized by various immune evasion strategies. These viruses have co-evolved with an increasingly sophisticated mammalian immune system which has continually placed pressure on their continued survival. This paper proposes that exposure to directly antiviral cytokines, namely TNF and members of the IFN family, during inflammatory and early immune responses, exerted particularly strong selective pressures on viruses, and has had a critical influence on the development of viral immune evasion strategies and pathogenesis. In the context of antiviral cytokine activity, this report concentrates on two DNA virus families with contrasting pathogenic and immune evasion strategies, namely poxviruses and HSV.

The effect of enhancing antibodies on TNF interactions with its specific receptor: consequences for in vitro TNF antiviral activity.

This study examines the interaction of TNF with its receptor(s) in the presence of antibodies which have been previously found to enhance the in vivo antiviral and antitumor activities of this cytokine. The presence of Mab 32 has been previously shown to enhance the binding of 125I-TNF to the surface L929 cells (13), and this property was also found in the present study for HeLa cells. In addition to this, Mab 32 was found to enhance the internalization of the TNF ligand into both L929 and HeLa cells compared to control treated cultures. The consequences of such enhanced TNF-binding on TNF antiviral activity were examined in both L929 cells and HeLa cells. It was discovered that the similarities in antibody-enhanced TNF binding and internalization contrasted dramatically with the sensitivities of these two cell lines to the antiviral actions of TNF, both with and without Mab 32 (viz., L929 cells were sensitive; HeLa cells were resistant). It has been proposed that the modulation of TNF-R expression, particularly by IFNs, is an important factor in TNF's biological effects. It has been shown that the presence of IFN-gamma, with TNF plus specific enhancing antibodies, further augmented antiviral activity in vivo (13). This finding stimulated interest in examining IFN-gamma modulation of TNF-R as a factor in the antiviral activity of TNF. The expression of TNF receptor(s) in TNF- and/or IFN-gamma-exposed cells, both with and without HSV-1 infection, was therefore examined. TNF alone could induce a dose-dependent increase in receptor expression which was not significantly increased by Mab 32. Exposure of L929 cells to IFN-gamma alone also induced TNF receptor expression in mock-infected cells. HSV-1 infection of L929 cells resulted in a significant upregulation of TNF-R expression which was reversed if the cells had been preexposed to IFN-gamma. The inclusion of TNF with IFN-gamma before infection restored TNF-R expression but did not show any further synergistic or additive effects on TNF-R expression. Some minor increases in TNF-R expression were seen if Mab 32 was included with these two cytokines.

The enhancement of human tumor necrosis factor-alpha antiviral activity in vivo by monoclonal and specific polyclonal antibodies.

This report describes the potentiation of the antiviral effects of human tumour necrosis factor-alpha (TNF-alpha) in vivo by specific antibodies. Complexes of TNF with either an anti-TNF monoclonal or polyclonal antibody at optimal doses induced a significantly greater antiviral (vaccinia virus) state in CBA/H mice than TNF alone. Furthermore, an antiviral synergy between murine gamma-interferon and TNF was found in vivo when the latter was in complex with enhancing antibody. Two other inbred mouse strains, C57/B6 and BALB/c, were also examined under these conditions. These antibodies greatly increase the binding of TNF to the surface of cells, which may account for the observed enhancement of TNF activity. Such antibodies may be of value clinically in viral diseases and cancer therapy where an increase in TNF activity, in the absence of side effects, would lead to more effective use of this cytokine in human therapy.