Unusual intranuclear filaments in the circulating lymphocytes of patients with multiple sclerosis and optic neuritis.

Mononuclear cells obtained from the peripheral blood of patients with multiple sclerosis (MS) and optic neuritis (ON) were examined by electron microscopy. Unusual intranuclear filaments identical with the so-called paramyxovirus-like filaments were found in circulating lymphocytes from 5 patients with MS and 3 with ON. They were observed in 0.1 to 0.5% of the mononuclear cells. Electron microscopy of the blood cells from 5 other patients with ON, 3 patients with neurologic diseases other than MS, and 4 normal subjects failed to reveal similar structures. The filaments were also absent from blood cells from blood cells from normal subjects during the process of autolysis.     

Virological and immunological indices in patients with multiple sclerosis

The level of specific antibodies to viruses of measles, parotitis, type-6 herpes, Epstein-Barr, tick-borne encephalitis and Borrelia burgdorferi as well as presence of genetic samples and antigens of the above infectious antigens were studied in patients with multiple sclerosis (MS). The cytokines Th1 and Th2 parameters were investigated in blood serum of patients at different MS stages. The titer of antibodies to measles virus was noted to be increasing in MS patients with age and disease aggravation. The level of antibodies to any of the studied infectious agents, except for the type-6 herpes virus, was not dynamically changing for as long as 9 months. The viral genetic samples (measles RNA) were detected just once in 2 patients; the detection time coincided in both cases with MS aggravation. The cytokines dynamics failed to correlate with MS aggravation or exacerbation while the total index of all studied cytokines was decreased. A high MMPw 9 content in blood serum correlated with MS exacerbation in 1 patient.     

Increased levels of antiviral MxA protein in peripheral blood of patients with a chronic disease of unknown etiology

Interferon alpha (IFN-alpha) is synthesized in response to viral infections. MxA protein, induced specifically by IFN-alpha and beta, expressed in peripheral blood cells, is detected more consistently than circulating IFN-alpha in serum of patients with viral infections. Thus, activation of the IFN-alpha/MxA system can be used as additional marker of the presence of a virus in patients. Therefore MxA protein and IFN-alpha levels were measured in patients with multiple sclerosis (MS), a chronic neurological disease of unknown etiology, in order to investigate the possible role of viruses in the expression of this disease. The means of MxA values obtained by using an immunochemiluminescent assay were significantly higher in blood of patients with remitting (n = 197) or relapsing (n = 39) multiple sclerosis (MS) patients and in patients with viral infections than in blood from healthy controls (n = 25) and from patients with bacterial infections (n = 12). Intra-individual variance in MxA levels in seven clinically stable remitting patients with MS was observed in the course of a follow-up, and high MxA levels were detected in three of them in blood samples collected consecutively over several months. By using an ultra sensitive assay, a higher MxA-inducer activity was obtained with sera from MS patients (n = 39) than with those from healthy controls (n = 12). Experiments with neutralizing antibodies proved that this activity in serum from patients was due to IFN-alpha, whereas IFN-alpha could not be detected by other methods. Altogether these results demonstrate that there is an activation of the IFN-alpha/MxA system in MS patients, which is consistent with the hypothesis that a viral infection may be associated with MS.     

Peripheral blood lymphocyte response to acute infections in humans.

The response of subpopulations of peripheral venous blood lymphocytes to systemic bacterial or viral infections was studied. B-lymphocytes were defined by the presence of surface binding sites for mu chains, which were determined by immunofluorescent staining. T-lymphocytes were defined by the ability to form active sheep cell rosettes. Virus-infectible lymphocytes, which may represent activated T-lymphocytes, were defined by the ability to support virus replication. Patients with bacterial infections had an increase in the B-lymphocytes of peripheral venous blood, whereas patients with viral infections had an increase in T-lymphocytes as compared to controls. The number of virus-infectible lymphocytes was increased in patients with bacterial infections but not in patients with viral infections. These studies suggest that subpopulations of human peripheral blood lymphocytes vary in response to different types of infectious agents.     

Use of polymerase chain reaction for the diagnosis of central nervous system infections

In the present work, the polymerase chain reaction (PCR) assay and his variants RT-PCR and Multiplex PCR were applied for the detection of specific sequences of Enterovirus, Human Herpes viruses (Herpes simple virus, Human Herpes virus type 6, Cytomegalovirus, Epstein Barr virus, and Varicella Zoster), Human Immunodeficiency virus, Toxoplasma gondii, Mycobacterium tuberculosis and Mycoplasma pneumoniae in patients' cohorts grouped by medical suspicion of meningoencephalitis. Of 326 samples of processed cerebrospinal fluid (CSF), 93 samples (28.5%) were positive for the different infectious agents. In the group of patients with clinical diagnosis of viral meningoencephalitis (n=212), there was obtained a whole of 73 positive samples (34.4%), of which 37 patients were positive to Enterovirus (50.7%), 19 were positive to VHS (26%) and 10 patients (13.7%) were positive to CMV. Other viral agents as VZV, EBV and HVH6 were detected in minor frequency. The 114 remaining samples were analyzed applying specific PCR to each pathogen for strict medical indication, being able to detect the presence of Human Immunodeficiency Virus (40%), Mycoplasma pneumoniae (40%), Toxoplasma gondii (14%) and Mycobacterium tuberculosis (12%) in CSF samples. The results obtained in this study demonstrate the convenience of the application of the molecular assays in the laboratory diagnosis of the meningoencefalitis of different etiology. Besides this, it is also a very valuable tool for the clinical management of the patients and for the execution of the epidemiological studies.     

T Cells Recognizing Multiple Peptides of Myelin Basic Protein are Found in Blood and Enriched in Cerebrospinal Fluid in Optic Neuritis and Multiple Sclerosis

The cause of multiple sclerosis (MS) is unknown. Recently reported abnormal T-cell responses to several myelin proteins and myelin basic protein (MBP) peptides in peripheral blood constitute one line of evidence that autoimmune mechanisms could be involved in the pathogenesis of the disease. Monosymptomatic unilateral optic neuritis (ON) is a common first manifestation of MS and important to examine for a possible restriction of the T-cell repertoire early in the disease. T-cell activities to MBP and the MBP amino acid sequences 63–88, 110–128 and 148–165 were examined by short-term cultures of mononuclear cells from cerebrospinal fluid (CSF) and blood in the presence of these antigens, and subsequent detection and counting of antigen-specific T cells that responded by interferon-gamma (IFN-γ) secretion. Most patients with MS and ON had MBP and MBP peptide-reactive T cells in CSF, amounting to mean values of between about 1 per 2000 and 1 per 7000 CSF cells and without immunodominance for any of the peptides. Numbers were 10-fold to 100-fold lower in the patients' blood. Values were similar in ON and MS, and no evidence was obtained for a more restricted T-cell repertoire in ON. The MBP peptide-recognizing T-cell repertoire was different in CSF than in blood in individual patients with ON and MS, thereby giving further evidence for an autonomy of the autoimmune T-cell response in the CSF compartment. No relations were observed between numbers of autoreactive T cells and presence of oligoclonal IgG bands in CSF or abnormalities on magnetic resonance imaging of the brain in ON or clinical variables of MS. The high numbers of MBP and MBP peptide-reactive T cells could play a role in the pathogenesis of ON via secretion of effector molecules, one of them being IFN-γ, as well as in the transfer of ON to MS.     

HIV excretion patterns and specific antibody responses in body fluids

HIV excretion patterns and specific antibody responses were evaluated in blood, semen, female genital secretions, saliva, and crevicular fluid. Samples were examined for infectious virus, viral antigens, viral nucleic acid, HIV specific IgG, IgA, anti-nef, and anti-p24. Viral load in peripheral blood appeared to increase with disease progression. The proportion of patients with antibody responses specific for nef and p24 was also lower among patients with more advanced disease. Infectious virus and viral antigens were detected infrequently and at lower levels in body fluids than in blood, which may reflect the presence of local antibodies. HIV nucleic acid was detected in some semen and saliva samples in the absence of infectious virus.     

Detection of viral DNA sequences in the cerebrospinal fluid of patients with multiple sclerosis

The role of viruses in the pathogenesis of multiple sclerosis (MS) is a subject of heated debate. The presence of six different neurotropic viruses was sought, including JC virus (JCV), varicella zoster virus (VZV), human herpesvirus 6 (HHV‐6), and Epstein‐Barr virus (EBV), in cerebrospinal fluid (CSF) samples collected from 51 patients with MS and 30 patients with other neurological diseases. Cell‐free or cell‐associated viral DNA in CSF samples was detected by real‐time PCR, and viral loads were determined. Magnetic resonance imaging (MRI) examinations were also performed to look for active lesions. Cell‐associated JCV DNA was detected in 3 of the 51 patients with MS and in 2 of the 30 patients with other neurological disease. Cell‐free JCV DNA was detected in one additional patient with MS. Cell‐free VZV DNA was detected in one patient without MS, cell‐free HHV‐6 was detected in one patient with MS, and cell‐free EBV was detected in one patient with MS. All other study patients had no detectable viral DNA in CSF samples and no double infections were found. The small percentage of patients with detectable viral DNA in CSF samples was comparable between patients with MS and those with other neurological disease, and presence of viral DNA was not a predictor of brain lesions. Additional observations suggest that cell trafficking from the periphery, rather than leakage through the blood–brain barrier, results in the transport of viruses to the CNS, where local immunosurveillance can control viral replication in immunocompetent individuals. J. Med. Virol. 82:1051–1057, 2010. © 2010 Wiley‐Liss, Inc.     

Enhanced detection of infectious airborne influenza virus

Current screening methodologies for detecting infectious airborne influenza virus are limited and lack sensitivity. To increase the sensitivity for detecting infectious influenza virus in an aerosol sample, the viral replication assay was developed. With this assay, influenza virus is first amplified by replication in Madin-Darby canine kidney (MDCK) cells followed by detection with quantitative PCR (qPCR). Spanning a 20-h replication period, matrix gene expression levels from infectious virus were measured at several time points using qPCR and found to exponentially increase. Compared with the traditional culture-based viral plaque assay, the viral replication assay resulted in a 4.6 × 10(5) fold increase in influenza virus detection. Furthermore, viral replication assay results were obtained in half the time of the viral plaque assay. To demonstrate that the viral replication assay is capable of detecting airborne influenza virus, dilute preparations of strain A/WS/33 were loaded into a nebulizer, aerosolized within a calm-air settling chamber and subsequently collected using NIOSH Two-Stage Bioaerosol Samplers. At the most diluted concentration corresponding to a chicken embryo infectious dose 50% endpoint (CEID(50)) of 2.8E+02/ml, the viral replication assay was able to detect infectious influenza virus that was otherwise undetectable by viral plaque assay. The results obtained demonstrate that the viral replication assay is highly sensitive at detecting infectious influenza virus from aerosol samples.     

Experience of detection of rubella markers during local outbreaks in West Siberia

This paper presents an analysis of samples from 75 patients for the presence of rubella virus, viral RNA, and specific antibodies. For all samples, RNA detection was higher than virus isolation. It was found that it was not possible to isolate rubella virus in every sixth clinical sample containing the viral RNA. Primary structures of the site (from the 8072nd to 8291st nucleotide) of the rubella virus genome from 14 samples were determined. This paper shows that all the samples of rubella virus belong to the first genotype, subgroups 1h, and very likely to subgroups 1a and 1F. For the first time, the circulation of rubella virus of the genotype has been shown both prior to the start of mass vaccination in Western Siberia and after.     

Pityriasis rosea – a virus-induced skin disease? An update

Summary.  Pityriasis rosea (PR) is an acute, inflammatory skin disease of unknown cause. Clinical and experimental findings indicate an infectious etiology of PR. Various infectious agents including viruses have been proposed as causative agents and their presence in PR samples has been extensively investigated. Recently, human herpesvirus 7 was linked to PR, but contradictory findings have been reported by various investigators. Here, we describe the features of PR that suggest an infectious cause and review the data from viral studies in PR reported in the literature. In addition, we present a pathogenetic model of PR which may be helpful in planning and evaluating studies for the search of a putative PR-associated virus. Based on the current state of knowledge, none of the known viruses could, so far, be conclusively associated with PR. Received February 22, 2000 Accepted March 17, 2000     

Detection of infectious agents by polymerase chain reaction in human aortic wall.

INTRODUCTION: Several studies have been suggested that infectious agents may induce or progress the process of atherosclerosis in humans. In the present study, the samples of visually healthy human aortic wall were examined for the presence of Chlamydia pneumoniae, Mycoplasma pneumoniae, Helicobacter pylori, herpes simplex virus (HSV), and cytomegalovirus (CMV). METHODS: Bacterial DNA of C. pneumoniae, M. pneumoniae, and H. pylori and viral DNA of HSV and CMV were analyzed by polymerase chain reaction. The specimens were obtained from 40 patients with atherosclerotic three-vessel stable coronary artery disease referred to surgical revascularization (coronary group) and 20 controls referred to aortic valve replacement (valve group). RESULTS: C. pneumoniae was detected in 11 of 40 samples of aorta in coronary group (27.5%) and 5 of 20 in valve group (25%). M. pneumoniae was found in 6 of 40 (15%) and 5 of 20 (25%) samples, and CMV was found in 22 of 40 (55%) and 10 of 20 (50%) samples. The most frequent detected pathogens were H. pylori and HSV. H. pylori was found in 32 of 40 samples of aortic wall in coronary group (80%) and 17 of 20 samples in valve group (85%), whereas HSV was found in 27 of 40 (67.5%) and 17 of 20 (85%) aortic wall specimens. CONCLUSION: Results demonstrate that C. pneumoniae, M. pneumoniae, H. pylori, CMV, and HSV can be detected in macroscopically healthy aortic wall of coronary and valve patients in similar frequency, which do not support hypothesis concerning the role of microorganisms in atherosclerosis etiology.     

Quantitation of cytomegalovirus DNA by blot hybridization in blood leukocytes of viremic patients

We describe a technique for quantitation of viral DNA in blood leukocytes during viremic infection with human cytomegalovirus (CMV). Using a cloned subgenomic DNA probe and a blot hybridization assay, small amounts of viral DNA within samples of leukocyte DNA could be quantitated reproducibly using a videodensitometer. Critical components of the assay were: (1) a direct relationship between optical density and known amounts of viral DNA diluted in cellular DNA as positive standard samples and, (2) determination of the proper duration of autoradiographic exposure. The technique was sufficiently sensitive to detect 10 picograms of CMV DNA in the presence of microgram quantities of host cell DNA. In addition, samples containing minute amounts of CMV DNA could reliably be distinguished from samples that were negative. We have used the technique to characterize the pathogenesis of CMV viremia and to monitor the effects of antiviral chemotherapy. This procedure could easily be applied to pathogenetic studies of a variety of other infectious agents.     

Advances in neuroimmunological laboratory studies on neuromuscular diseases

The recent methodological advances in molecular biology, immunology, and genetics have clarified neuroimmunological problems in axonal Guillain-Barré syndrome, seronegative myasthenia gravis, paraneoplastic neurologic syndromes and many others. In addition to clinical and serological studies in peripheral neuropathies, the origins and measurement of anti-ganglioside antibodies and relationships to similar carbohydrate structures on infectious organisms are discussed in the context of molecular mimicry hypothesis, especially related with both the localization of relevant gangliosides in the nerve and clinical phenotypes. Major advances have been made in animal modeling of anti-ganglioside antibody-associated disease. An explanation for muscular weakness in 10-15% of patients with seronegative myasthenia gravis who lack autoantibodies to acetylcholine receptors(AchRs) appears to be the autoantibodies to muscle-specific receptor tyrosine kinase (MuSK). MuSK mediates agrin-induced clustering of AchRs during synapse formation. These autoantibodies to the extracellular domain of MuSK inhibit its function in tissue culture. Isoelectric focusing (IEF) and agar gel electrophoresis (AGE) are used to examine cerebrospinal fluid (CSF) and sera from patients with multiple sclerosis (MS). The CSF oligoclonal IgG bands (OB) are less frequently observed in Japanese MS patients compared with Caucasian patients. Few optic-spinal form of MS (OS-MS) was positive for OB by agarose gel electrophoresis, but IEF is more sensitive than AGE. Recent IEF data revealed some OS-MS patients were positive for OB. The neuroimmunological advances revealed the relationship between the neuroimmunological diseases and the putative autoantibodies as diagnostic markers, for example, HAM and hnRNP-A1, MSand anti-hnRNP-B1 antibody, opsoclonus-myoclonus syndrome and anti-GluRdelta2 antibody, Rasmussen encephalitis and anti-GluR3 antibody, paraneoplastic brainstem encephalitis and anti-Ma2 antibody, and so on.     

Co-purification of soluble membrane cofactor protein (CD46) and human herpesvirus 6 variant A genome in serum from multiple sclerosis patients

The association of human herpesvirus 6 (HHV-6) and multiple sclerosis (MS) has been supported by several immunological and molecular studies. Recently, membrane cofactor protein (CD46) has been identified as the cellular receptor for the A and B variants of HHV-6. Elevated levels of soluble CD46 (sCD46) have been reported in the serum and CSF of MS patients. The aim of this study was to investigate a possible correlation between elevated levels of soluble CD46 and the presence of serum HHV-6 DNA in MS patients. An immunoaffinity column comprised of immobilized monoclonal antibodies to CD46 was developed to isolate sCD46 from cell free body fluids of MS patients and controls. After immunoaffinity purification, DNA was extracted from anti-CD46 column eluates and subjected to PCR amplification. Of the 42 MS samples tested, 4 serum samples were HHV-6 positive, 3 of which were typed as HHV-6A. The co-purification of sCD46 and HHV-6 DNA from MS sera indicates that HHV-6 is tightly connected to its receptor, CD46, in the serum of MS patients.     

Prevalence of primary versus reactivated Epstein-Barr virus infection in patients with VCA IgG-, VCA IgM- and EBNA-1-antibodies and suspected infectious mononucleosis.

BACKGROUND: In Epstein-Barr virus (EBV) infection, IgG- and IgM-antibodies to viral capsid antigen (VCA) and IgG-antibodies to Epstein-Barr nuclear antigen 1 (EBNA-1) can occur simultaneously both in late primary infection and during subclinical viral reactivation in immunocompetent persons, and the differential diagnosis is of importance. OBJECTIVES: To study the prevalence of primary infection and serological reactivation in patients with suspected primary EBV infection and with all three parameters present. STUDY DESIGN: Fifty serum samples from 43 consecutive patients referred for suspected infectious mononucleosis and positive for VCA IgG-, VCA IgM- and EBNA-1-antibodies by EIA, were tested for IgG-antibody avidity with an EBV IgG immunoblot. Sera were also tested for heterophile antibodies (HA). To verify the presence of IgM-antibodies an EBV IgM immunoblot was performed when high-avidity IgG-antibodies were found. RESULTS AND CONCLUSIONS: Of 43 patients with suspected primary EBV infection and VCA IgG-, VCA IgM- and EBNA-1-antibodies present, only 18 patients (42%) had a late primary infection. Twenty-one patients (49%) had high-avidity IgG-antibodies, indicating an IgM response due to reactivation, thus suggesting other causes for their symptoms. In 10 of these 21 patients the presence of IgM-antibodies was confirmed by immunoblot, indicating reactivation as a cause of IgM-antibodies in at least 23% of the 43 patients studied. Of 18 patients with primary infection, HA were detected in 16 (94%) of 17 patients tested. Only one (5%) of the patients with high-avidity antibodies had HA. Absence of HA in patients with this serological pattern is therefore a good indicator of reactivation, and conversely, the presence of HA is a good indicator of primary infection. In HA negative patients, avidity testing could be used for differential diagnosis.     

Detection of HBV infectivity by spot hybridization in HBeAg-negative chronic carriers: HBV DNA in sera from asymptomatic and symptomatic subjects

DNA of hepatitis B virus (HBV DNA) in sera from HBeAg-positive carriers is now the most important and reliable marker of infectivity, but its significance in the progression of chronic hepatitis in anti-HBe carrier status is still under discussion. In this study, viral DNA was tested by a simplified spot hybridization method in sera of 206 HBeAg-negative Italian subjects. In a group of 153 HBsAg carriers, we found that 15.6% of anti-HBe-positive and 10.5% of anti-HBe-negative samples contained viral DNA. No HBV DNA was revealed in 38 HBsAg-negative nor in 15 anti-HBs-positive subjects with different serological markers of HBV. Viral DNA in sera of HBeAg-negative patients with severe chronic liver disease was correlated with increased alaninetransferase activity and IgM anti-HBc. Thus the presence of HBV DNA in these sera not only predicts which subjects are potentially infectious but also indicates chronic progression of hepatitis. Finally, viral DNA extracted from Dane particles of nine anti-HBe-positive sera was characterized by the Southern blot technique. The hybridization pattern shows bands indicating the presence of replicative intermediates.