SARS-CoV-2 infection reduces quality of sperm parameters: prospective one year follow-up study in 93 patients.

BACKGROUND: Short- and long-term implications of SARS-CoV-2 on the quality of the sperm and the results of this on fertility remain largely unknown due to lack of longitudinal studies. In this longitudinal observational cohort study, we aimed to analyse the differential effect and the impact of SARS-CoV-2 infection on different semen quality parameters. METHODS: Sperm quality was assessed using the World Health Organization criteria, DNA damage to sperm cells by quantifying the DNA fragmentation index (DFI) and the high-density stainability (HDS), IgA- and IgG-anti-sperm antibodies (ASA) were assessed with light microscopy. FINDINGS: SARS-CoV-2 infection was associated with sperm parameters that were independent of spermatogenic cycle like progressive motility, morphology, DFI and HDS, as well as spermatogenic cycle dependent parameters such as sperm concentration. Detection of IgA- and IgG-ASA allowed classification of patients in three different groups according to its sequence of appearance in sperm during post-COVID-19 follow-up. The maximum progressive motility was lowest during follow-up in patients without ASA (41.9%), intermediate in patients with only IgA-ASA (46.2%) and highest inpatients who had both IgA- and IgG-ASA (54.9%). INTERPRETATION: SARS-CoV-2 infection was associated with changes of all analysed sperm parameters to a different degree which is also observed in their return to normality and is suggestive of individual variations in the patient's immune system performance. Firstly, sperm production is decreased through temporal immune mediated arrest of active meiosis, and secondly immune induced sperm DNA damage prevents fertilization if transferred to the oocyte. Both mechanisms are temporal, and most sperm parameters return to baseline after infection. FUNDING: AML (R20-014), Femicare.

Negative Impact of Elevated DNA Fragmentation and Human Papillomavirus (HPV) Presence in Sperm on the Outcome of Intra-Uterine Insemination (IUI).

We wanted to determine the sperm DNA fragmentation index (DFI) cutoff for clinical pregnancies in women receiving intra-uterine insemination (IUI) with this sperm and to assess the contribution of Human Papillomavirus (HPV) infection on sperm DNA damage and its impact on clinical pregnancies. Prospective non-interventional multi-center study with 161 infertile couples going through 209 cycles of IUI in hospital fertility centers in Flanders, Belgium. Measurement of DFI and HPV DNA with type specific quantitative PCRs (HPV 6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66 and 68) in sperm before its use in IUI. Clinical pregnancy (CP) rate was used as the outcome to analyze the impact on fertility outcome and to calculated the clinical cutoff value for DFI. A DFI criterion value of 26% was obtained by receiver operating characteristic (ROC) curve analysis. Couples with a male DFI > 26% had significantly less CPs than couples with DFI below 26% (OR 0.0326; 95% CI 0.0019 to 0.5400; p = 0.017). In sperm, HPV prevalence was 14.8%/IUI cycle. Sperm samples containing HPV had a significantly higher DFI compared to HPV negative sperm samples (29.8% vs. 20.9%; p = 0.011). When HPV-virions were present in sperm, no clinical pregnancies were observed. More than 1 in 5 of samples with normal semen parameters (17/78; 21.8%) had an elevated DFI or was HPV positive. Sperm DFI is a robust predictor of clinical pregnancies in women receiving IUI with this sperm. When DFI exceeds 26%, clinical pregnancies are less likely and in vitro fertilization techniques should be considered.

Infectious human papillomavirus virions in semen reduce clinical pregnancy rates in women undergoing intrauterine insemination.

OBJECTIVE: To study the influence of human papillomavirus (HPV) virions present in different sperm fractions of male partners of women undergoing IUI on fertility outcome. DESIGN: Prospective noninterventional multicenter study. SETTING: Inpatient hospital fertility centers. PATIENT(S): Seven hundred thirty-two infertile couples undergoing 1,753 IUI cycles with capacitated sperm. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Biochemical and clinical pregnancy rate in IUI cycles with HPV-positive or HPV-negative semen. RESULT(S): Five hundred seventy-three infertile couples undergoing 1,362 IUI cycles were enrolled. Work-up of the 1,362 sperm samples that were used for IUI generated 3,444 separate sperm fractions. Each of the sperm fractions was tested with quantitative polymerase chain reaction for 18 different HPV types (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, and 68). HPV prevalence in sperm was 12.5%/IUI cycle. When infectious HPV virions were detected in sperm, a significant decrease in clinical pregnancies was observed when compared with HPV-negative cycles (2.9% vs. 11.1 %/cycle). Above a ratio of 0.66 HPV virions/spermatozoon no pregnancies occurred (sensitivity 100%, specificity 32.5%). CONCLUSION(S): Women inseminated with HPV-positive sperm had 4 times fewer clinical pregnancies compared with women who had HPV-negative partners. Detection of HPV virions in sperm is associated with a negative IUI outcome and should be part of routine examination and counseling of infertile couples. EUROPEAN CLINICAL TRIALS DATABASE NUMBER: 2017-004791-56.

Human Papillomavirus Positivity in Women Undergoing Intrauterine Insemination Has a Negative Effect on Pregnancy Rates.

BACKGROUND: Sexually transmitted infections are a major cause of infertility. Human papillomavirus (HPV) infection is one of the most common viral infections of the female genital tract. Only a limited number of studies have investigated the influence of HPV on fertility and its impact remains controversial. OBJECTIVE: We investigated the relationship between cervical HPV infection and pregnancy outcome after intrauterine insemination (IUI). Since other sexually transmitted infections could also influence outcome, we also analyzed the influence of Trichomonas vaginalis (TV) and Chlamydia trachomatis (CT) on pregnancy outcome. METHODS: We performed a retrospective analysis of 590 women who underwent 1,529 IUI cycles at AML between 2010 and 2014. Positivity of 18 different HPV types (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68) and TV was assessed by PCR in cervical cytology specimens. CT status was ascertained by detection of IgA/IgG antibodies on serum samples or by PCR on cervical swabs. RESULTS: The HPV prevalence per IUI cycle was 11.0 and 6.9% for CT; none of the women tested positive for TV. HPV-positive women were six times less likely to become pregnant after IUI (1.87 vs. 11.36%; p = 0.0041). There was no significant difference in pregnancy rates between women with or without a history of CT (8.51 vs. 11.10%; p > 0.05). CONCLUSION: Detection of HPV is associated with a negative IUI outcome.

Increased expression of activation antigens on CD8+ T lymphocytes in Myalgic Encephalomyelitis/chronic fatigue syndrome: inverse associations with lowered CD19+ expression and CD4+/CD8+ ratio, but no associations with (auto)immune, leaky gut, oxidative and nitrosative stress biomarkers.

BACKGROUND: There is now evidence that specific subgroups of patients with Myalgic Encephalomyelitis / chronic fatigue syndrome (ME/CFS) suffer from a neuro-psychiatric-immune disorder. This study was carried out to delineate the expression of the activation markers CD38 and human leukocyte antigen (HLA) DR on CD4+ and CD8+ peripheral blood lymphocytes in ME/CFS. METHODS: Proportions and absolute numbers of peripheral lymphocytes expressing CD3+, CD19+, CD4+, CD8+, CD38+ and HLA-DR+ were measured in ME/CFS (n=139), chronic fatigue (CF, n=65) and normal controls (n=40). RESULTS: The proportions of CD3+, CD8+, CD8+CD38+ and CD8+HLA-DR+ were significantly higher in ME/CFS patients than controls, while CD38+, CD8+CD38+, CD8+HLA-DR+ and CD38+HLA-DR+ were significantly higher in ME/CFS than CF. The percentage of CD19+ cells and the CD4+/CD8+ ratio were significantly lower in ME/CFS and CF than in controls. There were highly significant inverse correlations between the increased expression of CD38+, especially that of CD8+CD38+, and the lowered CD4+/CD8+ ratio and CD19+ expression. There were no significant associations between the flow cytometric results and severity or duration of illness and peripheral blood biomarkers of oxidative and nitrosative stress (O&NS, i.e. IgM responses to O&N modified epitopes), leaky gut (IgM or IgA responses to LPS of gut commensal bacteria), cytokines (interleukin-1, tumor necrosis factor-α), neopterin, lysozyme and autoimmune responses to serotonin. CONCLUSIONS: The results support that a) increased CD38 and HLA-DR expression on CD8+ T cells are biomarkers of ME/CFS; b) increased CD38 antigen expression may contribute to suppression of the CD4+/CD8+ ratio and CD19+ expression; c) there are different immune subgroups of ME/CFS patients, e.g. increased CD8+ activation marker expression versus inflammation or O&NS processes; and d) viral infections or reactivation may play a role in a some ME/CFS patients.

Lower whole blood glutathione peroxidase (GPX) activity in depression, but not in myalgic encephalomyelitis / chronic fatigue syndrome: another pathway that may be associated with coronary artery disease and neuroprogression in depression.

BACKGROUND: Major depression and myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) are two disorders accompanied by an upregulation of the inflammatory and oxidative and nitrosative (IO&NS) pathways and a decreased antioxidant status. Moreover, depression is accompanied by disorders in inflammatory and neuroprogressive (IN-PRO) pathways. METHODS: This study examines whole blood glutathione peroxidase (GPX) in depression and in ME/CFS; GPX is an enzyme that reduces hydroperoxides by oxidizing glutathione and consequently protects the cells from oxidative damage. Blood was sampled in 39 patients with depression, 40 patients with ME/CFS and 24 normal volunteers. Whole blood was analysed for GPX activity using the Ransel assay (Randox). Severity of illness was measured by means of the Hamilton Depression Rating Scale (HDRS) and the Fibromyalgia and Chronic Fatigue Syndrome Rating Scale (FF scale). RESULTS: We found that whole blood GPX activity was significantly (p=0.001) lower in depressed patients than in normal controls and that there were no significant differences between ME/CFS and controls. In depression and ME/CFS, there were significant and inverse relationships between GPX activity and the FF items, depressed mood and autonomic symptoms. In depression, there were significant and negative correlations between whole blood GPX and the HDRS score and autonomic symptoms. DISCUSSION: The results show that lowered whole blood GPX activity contributes to the lowered antioxidant status in depression. Since GPX activity is a predictor of neuroprogression and coronary artery disease (CAD), lowered GPX activity in depression contributes to the IN-PRO pathways and the comorbidity between depression and CAD. Our results suggest that patients with depression would benefit from Ebselen or a supplementation with glutathione, N-Acetyl-l-Cysteine and selenium.

Increased plasma peroxides as a marker of oxidative stress in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS).

BACKGROUND: There is evidence that myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by activation of immune, inflammatory, oxidative and nitrosative stress (IO&NS) pathways. The present study was carried out in order to examine whether ME/CFS is accompanied by increased levels of plasma peroxides and serum oxidized LDL (oxLDL) antibodies, two biomarkers of oxidative stress. MATERIAL/METHODS: Blood was collected from 56 patients with ME/CFS and 37 normal volunteers. Severity of ME/CFS was measured using the Fibromyalgia and Chronic Fatigue Syndrome (FF) Rating Scale. RESULTS: Plasma peroxide concentrations were significantly higher in patients with ME/CFS than in normal controls. There was a trend towards significantly higher serum oxLDL antibodies in ME/CFS than in controls. Both biomarkers contributed significantly in discriminating between patients with ME/CFS and normal controls. Plasma peroxide and serum oxLDL antibody levels were both significantly related to one of the FF symptoms. CONCLUSIONS: The results show that ME/CFS is characterized by increased oxidative stress.

Comparison of three automated immunoassay methods for the determination of Epstein-Barr virus-specific immunoglobulin M.

In this study we compared the performances of three commercially available Epstein-Barr virus (EBV) immunoglobulin M (IgM) assays on highly automated immunoassay platforms: BioPlex 2200 (Bio-Rad Laboratories), Immulite 2000 (Siemens Healthcare Diagnostics), and Liaison (DiaSorin). As a confirmatory method, immunoblotting was performed. The specificity of the three EBV IgM assays was evaluated by testing 293 selected sera from patients with various infectious and noninfectious diseases. After the exclusion of 30 samples, the specificities were 96.2% for Liaison, 98.1% for Immulite, and 97.0% for BioPlex. For evaluation of the sensitivity, samples from 70 consecutive patients with a positive heterophile antibody test were examined, irrespective of clinical or biological findings. After the exclusion of six samples, the sensitivities were 89.1% for Liaison, 84.4% for Immulite, and 89.1% for BioPlex. Finally, in a prospective study performed with 500 samples obtained from consecutive patients and sent in by general practitioners, we also determined Epstein-Barr nuclear antigen IgG and viral capsid antigen IgG in a two-phase approach. Concordance of the EBV serologic status was 96.2% between Liaison and Immulite, 96.4% between Immulite and BioPlex, and 97.8% between BioPlex and Liaison. The three EBV IgM immunoassays that we evaluated have acceptable and comparable performances.

Increased plasma peroxides and serum oxidized low density lipoprotein antibodies in major depression: markers that further explain the higher incidence of neurodegeneration and coronary artery disease.

BACKGROUND: Major depression is characterized by a decreased antioxidant status, an induction of the inflammatory and oxidative and nitrosative (IO&NS) pathways and inflammatory-neurodegenerative (I&ND) pathways. This study examines two markers of oxidative stress in depression, i.e. plasma peroxides and serum oxidized LDL (oxLDL) antibodies. METHODS: Blood was sampled in 54 patients with major depression (mean+/-SD age=43.5+/-11.6 years) and 37 normal volunteers (43.6+/-11.1 years). The severity of illness was measured by means of the Hamilton Depression Rating Scale. The Fibromyalgia and Chronic Fatigue Syndrome Rating Scale was used to measure severity of "psychosomatic" symptoms in depression. RESULTS: We found significantly higher plasma peroxides (p=0.002) and serum oxLDL antibodies (p=0.0002) in depressed patients as compared to normal controls. There was no significant correlation between both markers and both independently from each other predicted major depression. There were significant correlations between the oxLDL antibodies and the scores on two items of the FF scale, i.e. gastro-intestinal symptoms and headache. DISCUSSION: The results show that major depression is accompanied by increased oxidative stress and lipid peroxidation. These results further extend the IO&NS pathophysiology of major depression. Since increased peroxides and oxLDL antibodies are predictors of coronary artery disease (CAD) and neurodegeneration, our findings suggest that IO&NS pathways are involved in the increased incidence of both CAD and neurodegeneration in depression.

Increased 8-hydroxy-deoxyguanosine, a marker of oxidative damage to DNA, in major depression and myalgic encephalomyelitis / chronic fatigue syndrome.

BACKGROUND: There is now evidence that major depression and myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) are accompanied by partially overlapping pathophysiological mechanisms, i.e. activation of various inflammatory and oxidative & nitrosative (IO&NS) pathways. OBJECTIVE: The aim of the present study was to examine the urinary excretion of 8-hydroxy-deoxyguanosine (8-OhdG), a marker of oxidative damage to DNA, in depression; ME/CFS; and depression and ME/CFS. METHODS: Toward this end, morning urine was sampled for the assays of 8-OHdG and creatinine, in 44 patients with ME/CFS; 25 with major depression; 23 with depression and ME/CFS; and 17 normal controls. Severity of fatigue and somatic symptoms was measured by means of the Fibromyalgia and CFS Rating (FF) scale. RESULTS: We found that 49.0% of the variance in the urinary excretion of 8-OHdG was predicted by the regression on creatinine. Consequently, the urinary 8-OHdG excretion should be expressed as the residualized 8-OHdG values after partialling out the effects of creatinine and not by computing the 8-OHdG / creatinine ratio. We found that the residualized urinary excretion of 8-OHdG (adjusted for creatinine) was significantly higher in patients with depression and ME/CFS than in normal controls and all other patients. In the patient group, there were significant correlations between the urinary 8-OHdG and the total score on the FF scale and sadness and flu-like malaise. CONCLUSIONS: The findings show increased oxidatively generated DNA damage in patients with major depression and ME/CFS and, therefore, further extent the role played by IO&NS pathways in the pathophysiology of both disorders. Since oxidatively damage to DNA is a risk factor for atherosclerosis and neurodegeneration, our results also explain previous findings on increased cardiovascular morbidity in depression and ME/CFS, and neurodegenerative processes in depression.

Coenzyme Q10 deficiency in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is related to fatigue, autonomic and neurocognitive symptoms and is another risk factor explaining the early mortality in ME/CFS due to cardiovascular disorder.

INTRODUCTION: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a medical illness characterized by disorders in inflammatory and oxidative and nitrosative (IO&NS) pathways. METHODS: This paper examines the role of Coenzyme Q10 (CoQ10), a mitochondrial nutrient which acts as an essential cofactor for the production of ATP in mitochondria and which displays significant antioxidant activities. Plasma CoQ10 has been assayed in 58 patients with ME/CFS and in 22 normal controls; the relationships between CoQ10 and the severity of ME/CFS as measured by means of the FibroFatigue (FF) scale were measured. RESULTS: Plasma CoQ10 was significantly (p=0.00001) lower in ME/CFS patients than in normal controls. Up to 44.8% of patients with ME/CFS had values beneath the lowest plasma CoQ10 value detected in the normal controls, i.e. 490 microg/L. In ME/CFS, there were significant and inverse relationships between CoQ10 and the total score on the FF scale, fatigue and autonomic symptoms. Patients with very low CoQ10 (<390 microg/L) suffered significantly more from concentration and memory disturbances. DISCUSSION: The results show that lowered levels of CoQ10 play a role in the pathophysiology of ME/CFS and that symptoms, such as fatigue, and autonomic and neurocognitive symptoms may be caused by CoQ10 depletion. Our results suggest that patients with ME/CFS would benefit from CoQ10 supplementation in order to normalize the low CoQ10 syndrome and the IO&NS disorders. The findings that lower CoQ10 is an independent predictor of chronic heart failure (CHF) and mortality due to CHF may explain previous reports that the mean age of ME/CFS patients dying from CHF is 25 years younger than the age of those dying from CHF in the general population. Since statins significantly decrease plasma CoQ10, ME/CFS should be regarded as a relative contraindication for treatment with statins without CoQ10 supplementation.

Acute parvovirus B19 infection frequently causes false-positive results in Epstein-Barr virus- and herpes simplex virus-specific immunoglobulin M determinations done on the Liaison platform.

During an outbreak of parvovirus B19 we collected serum samples from 68 nonpregnant patients in the region of Antwerp (Belgium). Fifty-seven (84%) of the parvovirus B19 immunoglobulin M (IgM)-positive sera had a positive result for Epstein-Barr virus (EBV) IgM by Liaison testing, 61 (90%) had a positive result for herpes simplex virus (HSV) IgM, 20 (29%) samples were positive for cytomegalovirus IgM, and 15 (22%) had a positive result for Borrelia burgdorferi sensu lato IgM. As assay interference was suspected, sera were further investigated by using additional infectious-disease serology tests and by performing various interference elimination procedures. We could show that the EBV IgM and HSV IgM results were false positives due to aspecific IgM reactions with the solid phase. All samples were also analyzed by a modified Liaison EBV IgM assay, based on the addition of polyvinylpyrrolidone and polyvinyl alcohol to the dilution buffer, which partially eliminated this type of assay interference. Although the Liaison is a very convenient, automated immunoassay platform, this study demonstrates the potential for improvement of mainly the EBV IgM and HSV IgM tests.

Prevention of assay interference in infectious-disease serology tests done on the liaison platform.

Immunoassay interference causing unexpected reactive results in magnetic-microparticle-based assays was detected. A systematic evaluation of Liaison Epstein-Barr virus immunoglobulin M showed that 5% of the positive results (0.4% of tested samples) could be explained by such interference. Adding chemical blocking reagents (polyvinylpyrrolidone and polyvinyl alcohol) to the assay buffers partially prevented this phenomenon.

Why fish oils may not always be adequate treatments for depression or other inflammatory illnesses: docosahexaenoic acid, an omega-3 polyunsaturated fatty acid, induces a Th-1-like immune response.

BACKGROUND: We have shown that a depletion of omega3 polysaturated fatty acids (PUFAs) plays a role in the pathophysiology of depression, in part because omega3 PUFAs have anti-inflammatory effects. omega3 PUFAs are frequently employed to treat depression. Most if not all antidepressants have negative immunoregulatory effects by decreasing the production of proinflammatory cytokines, such as interferon-gamma (IFNgamma) and/or increasing that of anti-inflammatory cytokines, such as interleukin10 (IL-10). AIM: The aim of the present study was to examine the immunoregulary effects of the omega3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and the omega6 PUFA, arachidonic acid (AA), on the production of interferon-gamma (IFNgamma), interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNFalpha). METHODS: This study examines the ex vivo effects of EPA (4.5 microM, 9 microM, 18 microM and 45 microM), DHA (1.3 microM, 3 microM, 6 microM and 13 microM) and AA (8 microM, 16 microM, 32 microM and 80 microM) on the LPS + PHA-stimulated production of IFNgamma, IL-10 and TNFalpha, and on the IFNgamma/IL-10 production ratio. RESULTS: We found that EPA did not have any significant effects on the above cytokines. DHA significantly increased the IFNgamma/IL-10 production ratio, caused by a greater reduction in IL-10 than in IFNgamma. AA significantly decreased TNFalpha production. DISCUSSION: The results show that DHA induces a Th-1-like immune response and that AA has anti-inflammatory effects by decreasing the production of TNFalpha. Thus, the immune effects of omega3 PUFAs are not compatible with what is expected from antidepressive substances. The results of the present study show that treatment with fish oils, containing DHA, should be avoided in the treatment of depression. Toward this end, highly concentrated and pure EPA seems to be indicated.

The immune effects of TRYCATs (tryptophan catabolites along the IDO pathway): relevance for depression - and other conditions characterized by tryptophan depletion induced by inflammation.

Immune activation is accompanied by induction of indoleamine (2,3)-dioxygenase (IDO), an enzyme which degrades tryptophan, a phenomenon which plays a role in the pathophysiology of major depression and post-natal depression and anxiety states. TRYCATs - tryptophan catabolites along the IDO pathway - such as kynurenine, kynurenic acid, xanthurenic acid, and quinolinic acid, have multiple effects, e.g. apoptotic, anti- versus pro-oxidant, neurotoxic versus neuroprotective, and anxiolytic versus anxiogenic effects. The aim of the present study was to study the immune effects of the above TRYCATS. Toward this end we examined the effects of the above TRYCATs on the LPS + PHA-induced production of interferon-gamma (IFNgamma), interleukin-10 (IL-10), and tumor necrosis factor-alpha (TNFalpha) in 18 normal volunteers. We found that the production of IFNgamma was significantly decreased by all 4 catabolites. Xanthurenic acid and quinolinic acid decreased the production of IL-10. Kynurenine, kynurenic acid, and xanthurenic acid, decreased the IFNgamma/IL-10 production ratio, whereas quinolinic acid increased this ratio. Kynurenic acid significantly reduced the stimulated production of TNFalpha. It is concluded that kynurenine, kynurenic acid, and xanthurenic acid have anti-inflammatory effects trough a reduction of IFNgamma, whereas quinolinic acid has pro-inflammatory effects in particular via significant decreases in IL-10. Following inflammation-induced IDO activation, some TRYCATs, i.e. kynurenine, kynurenic acid, and xanthurenic acid, exert a negative feedback control over IFNgamma production thus downregulating the initial inflammation, whereas an excess of quinolinic acid further aggravates the initial inflammation.

Decreased expression of CD69 in chronic fatigue syndrome in relation to inflammatory markers: evidence for a severe disorder in the early activation of T lymphocytes and natural killer cells.

There is some evidence that patients with chronic fatigue syndrome (CFS) suffer from immune abnormalities, such as immune activation and decreased immune cell responsivity upon polyclonal stimili. This study was designed to evaluate lymphocyte activation in CFS by using a CD69 expression assay. CD69 acts as a costimulatory molecule for T- and natural killer (NK) cell activation. We collected whole blood from CFS patients, who met CDC criteria, and healthy volunteers. The blood samples were stimulated with mitogens during 18 h and the levels of activated T and NK cells expressing CD69 were measured on a Coulter Epics flow cytometer using a three color immunofluorescence staining protocol. The expression of the CD69 activation marker on T cells (CD3+, CD3+CD4+, and CD3+CD8+) and on NK cells (CD45+CD56+) was significantly lower in CFS patients than in healthy subjects. These differences were significant to the extent that a significant diagnostic performance was obtained, i.e. the area under the ROC curve was around 89%. No differences either in the number of leukocytes or in the number or percentage of lymphocytes, i.e. CD3, CD4, CD8 and CD19, could be found between CFS patients and the controls. Patients with CFS show defects in T- and NK cell activation. Since induction of CD69 surface expression is dependent on the activation of the protein kinase C (PKC) activation pathway, it is suggested that in CFS there is a disorder in the early activation of the immune system involving PKC.

Not in the mind but in the cell: increased production of cyclo-oxygenase-2 and inducible NO synthase in chronic fatigue syndrome.

Chronic fatigue syndrome (CFS) is a medically unexplained disorder, characterized by profound fatigue, infectious, rheumatological and neuropsychiatric symptoms. There is, however, some evidence that CFS is accompanied by signs of increased oxidative stress and inflammation in the peripheral blood. This paper examines the role of the inducible enzymes cyclo-oxygenase (COX-2) and inducible NO synthase (iNOS) in the pathophysiology of CFS. Toward this end we examined the production of COX-2 and iNOS by peripheral blood lymphocytes (PBMC) in 18 CFS patients and 18 normal volunteers and examined the relationships between those inflammatory markers and the severity of illness as measured by means of the FibroFatigue scale and the production of the transcription factor nuclear factor kappa beta (NFkappabeta). We found that the production of COX-2 and iNOS was significantly higher in CFS patients than in normal controls. There were significant and positive intercorrelations between COX-2, iNOS and NFkappabeta and between COX-2 and iNOS, on the one hand, and the severity of illness, on the other. The production of COX-2 and iNOS by PBMCs was significantly related to aches and pain, muscular tension, fatigue, concentration difficulties, failing memory, sadness and a subjective experience of infection. The results suggest that a) an intracellular inflammatory response in the white blood cells plays an important role in the pathophysiology of CFS; b) the inflammatory response in CFS is driven by the transcription factor NFkappabeta; c) symptoms, such as fatigue, pain, cognitive defects and the subjective feeling of infection, indicates the presence of a genuine inflammatory response in CFS patients; and d) CFS patients may be treated with substances that inhibit the production of COX-2 and iNOS.

Not in the mind of neurasthenic lazybones but in the cell nucleus: patients with chronic fatigue syndrome have increased production of nuclear factor kappa beta.

There is now some evidence that chronic fatigue syndrome is accompanied by an activation of the inflammatory response system and by increased oxidative and nitrosative stress. Nuclear factor kappa beta (NFkappabeta) is the major upstream, intracellular mechanism which regulates inflammatory and oxidative stress mediators. In order to examine the role of NFkappabeta in the pathophysiology of CFS, this study examines the production of NFkappabeta p50 in unstimulated, 10 ng/mL TNF-alpha (tumor necrosis factor alpha) and 50 ng/mL PMA (phorbolmyristate acetate) stimulated peripheral blood lymphocytes of 18 unmedicated patients with CFS and 18 age-sex matched controls. The unstimulated (F=19.4, df=1/34, p=0.0002), TNF-alpha-(F=14.0, df=1/34, p=0.0009) and PMA-(F=7.9, df=1/34, p=0.008) stimulated production of NFkappabeta were significantly higher in CFS patients than in controls. There were significant and positive correlations between the production of NFkappabeta and the severity of illness as measured with the FibroFatigue scale and with symptoms, such as aches and pain, muscular tension, fatigue, irritability, sadness, and the subjective feeling of infection. The results show that an intracellular inflammatory response in the white blood cells plays an important role in the pathophysiology of CFS and that previous findings on increased oxidative stress and inflammation in CFS may be attributed to an increased production of NFkappabeta. The results suggest that the symptoms of CFS, such as fatigue, muscular tension, depressive symptoms and the feeling of infection reflect a genuine inflammatory response in those patients. It is suggested that CFS patients should be treated with antioxidants, which inhibit the production of NFkappabeta, such as curcumin, N-Acetyl-Cysteine, quercitin, silimarin, lipoic acid and omega-3 fatty acids.

Rheumatoid factor interference in the determination of carbohydrate antigen 19-9 (CA 19-9).

BACKGROUND: Investigation of a 61-year-old Caucasian male suffering from fatigue and weight loss led to the finding of a carbohydrate antigen 19-9 (CA 19-9) concentration of 80 kU/L using an ADVIA Centaur analyser. Determination of CA 19-9 on Vidas, AxSYM and Architect i2000 systems gave normal results. His rheumatoid factor concentration was very high (900 kIU/L) and assay interference was suspected. METHODS: Besides using several laboratory procedures to show the cause of the interference, we tried to estimate the frequency of the suspected interference. Therefore, two studies were performed. The first was carried out in a multicentre setting using four different CA 19-9 methods on 51 randomly selected samples with high rheumatoid factor concentrations and ten samples containing no or very low rheumatoid factor. In the second study we used heterophilic blocking tubes for 68 routinely analysed samples with CA 19-9 concentrations ranging between 37 and 250 kU/L using an ADVIA Centaur analyser. RESULTS: In the multicentre study we found eight discrepant CA 19-9 results, but only one was clearly due to interference. We showed that the interference detected, just as in the index case, was caused by rheumatoid factor. The other discrepancies could not be explained, but are probably related to method-dependent differences. In the 68 routinely analysed samples, no interference could be shown using the heterophilic blocking tubes. CONCLUSIONS: Although interferences in the CA 19-9 assay are not frequent, the ADVIA Centaur system appears to be more sensitive to rheumatoid factor interference. The lack of standardisation remains an important issue for this assay. The determination of CA 19-9 during the follow-up of patients should be performed using a single method. If, however, there is any clinical doubt about a result, CA 19-9 should be determined using another method to exclude possible interferences.

Engineering camel single-domain antibodies and immobilization chemistry for human prostate-specific antigen sensing.

The specificity and affinity characteristics of antibodies make them excellent probes in biosensor applications. Unfortunately, their large size, unstable behavior, and random immobilization properties create numerous problems. The single-domain antigen-binding fragment derived from heavy-chain antibodies of camelids (termed VHH) offers special advantages in terms of size, stability, and ease of generating different antibody constructs. In this study, we show the potential of those VHHs in sensing human prostate-specific antigen (hPSA) by SPR technology. Different VHH constructs were immobilized onto commercial and custom-built sensor surfaces by metal chelation, biotin-streptavidin interaction, or covalent coupling. The detection of subnanogram per milliliter hPSA concentrations could be attained on a covalently coupled three-dimensional dextran surface. Moreover, the ratio of different hPSA isoform concentrations could be assessed via a sandwich assay and resulted in the detection of clinically significant antigen concentrations within 15 min. In addition, for the first time, the intrinsic protein stability is presented as an important probe design factor, since our results reveal that higher intrinsic stability offers higher resistance to harsh regeneration conditions. In conclusion, we present VHHs as a novel class of biosensor probes rivaling conventional antibodies and their derived antibody fragments.