Oligoclonal measles virus-specific IgG antibodies isolated from cerebrospinal fluids, brain extracts, and sera from patients with subacute sclerosing panencephalitis and multiple sclerosis

Measles virus-specific antibodies were isolated from sera, cerebrospinal fluids (CSF), and brain extracts of patients with subacute sclerosing panencephalitis (SSPE) and multiple sclerosis (MS) by absorption with measles antigens and subsequent acid elution of the antigen-antibody precipitates. Electrophoretically homogeneous measles antibodies were isolated from CSF or brain extracts in five patients with SSPE and in five out of seven patients with MS. Homogeneous IgG antibodies were also demonstrated in the sera from all SSPE patients and from three of the MS patients. The antibodies isolated from various control sera and from pooled CSF were electrophoretically heterogeneous. The results support the concept of a local synthesis in the nervous system of oligoclonal IgG antibodies to measles virus in all patients with SSPE and in some patients with MS. In SSPE, most or all oligoclonal IgG proteins of the CSF or brain carry measles antibody activities. In MS, only part of the oligoclonal IgG appears to be associated with measles antibody activity.     

Cell-mediated cytotoxicity against measles virus in SSPE. II. Analysis of cytotoxic effector cells

An analysis of subacute sclerosing panencephalitis (SSPE) lymphocytes was performed in order to find out whether T or K cells were involved in killing of 51Cr-labeled allogeneic measles virus-infected target cells. Lymphocyte donors were three patients with SSPE, 10 measles seropositive controls and 2 children with measles rash. It was found that about 75% of measles-specific cytotoxicity was lost after removal of Fc-receptor-bearing cells by adsorption onto immune complex monolayers. K cell activity (as measured by lysis of 51Cr-labeled-sensitized chicken red blood cells) was reduced to the same extent. After adsorption, the enhancing effect by specific antibody was no longer observed. Unfractionated peripheral lymphoid cells that had been treated with pronase and kept in culture 24 hr were inactive in the cytotoxicity test when compared to freshly isolated cells. However, cytotoxicity could be restored almost completely by the addition of measles antibodies. The results indicate that measles-specific cytotoxicity by peripheral lymphoid cells from all three groups of donors is mediated by K cells. It is probable that specificity is provided by a small amount of contaminating serum antibody or immune complexes.     

The detection of intrathecal synthesis of anti-herpes simplex IgG antibodies: comparison between an antigen-mediated immunoblotting technique and antibody index calculations. European Union Concerted Action on Virus Meningitis and Encephalitis

The detection of intrathecal antibody synthesis was compared by the calculation of antibody indices (AI) derived from ELISA techniques with the detection of virus-specific oligoclonal IgGs by an antigen-mediated capillary blot technique. Twenty-seven paired serum and cerebrospinal fluid (CSF) samples were examined from 15 immunocompetent patients with herpes simplex virus encephalitis (HSE) diagnosed by PCR on early CSF samples. These techniques were also applied to paired samples from 20 multiple sclerosis (MS) patients, 10 patients with other inflammatory neurological diseases and 10 patients with non inflammatory neurological disorders. There was a good correlation between the results obtained by AI and those obtained by immunoblotting, especially in HSE (2 discordant results out of 27). Discrepancies were more frequent (25%) in MS patients where a "polyspecific" reaction characterized by low affinity antibodies is known to occur. Some of the discrepancies could, in part, be due to serological cross-reaction with varicella zoster virus.     

A case of chronic encephalitis due to double infection with herpes simplex and measles viruses

In a healthy 49-year-old man, a decrease in job efficiency was noticed along with bizarre behavior. On admission, he was euphoric, childish, superficial and had increased libido. Neurological findings were normal. There were no abnormal findings on routine blood tests, hematochemistry or urine analysis. MRI showed no abnormal findings. However, single photon emission CT (SPECT) showed diffuse hypoaccummulation of tracer from the temporal to frontal regions. Lumbar puncture showed clear cerebrospinal fluid (CSF) with pleocytosis and an elevated protein level. Moreover, antibody IgG titers to herpes simplex virus (HSV) and measles virus were elevated, according to EIA [serum HSV -1,202.2x, measles virus 47.1x: CSF HSV-116.1x, measles virus 9.9x]. The ratio of serum to CSF antibody titers of HSV and measles virus were 12.5 and 4.75, respectively. The antibody index values of HSV and measles virus IgG titers were 8.42 and 22.22. The ratio of albumin was 105.7. Chronic, progressive HSV encephalitis is rare, and there have been very few reports of encephalitis due to double infection by HSV and another virus. Our patient was diagnosed as having encephalitis due to double infection with HSV and measles virus, because the ratio of serum to CSF antibody titers was less than 20 and the antibody index values were over 1.91. Moreover, since the IgG index was elevated and the ratio of albumin was not low, it was suggested that the blood-brain-barrier had not been disrupted, and antibodies were being produced chronically in the medullary cavity. Hyperaccummulation of tracer on SPECT studies has been reported in the early stages of HSV encephalitis. In our case, while CT and MRI showed no abnormal findings, SPECT showed diffuse hypoaccummulation. SPECT appears to be a useful tool in the diagnosis of this disorder. In case of chronic, progressive personality change in middle-aged adults, we must be aware of double virus infection of the brain as a possible causal factor.     

Immunologic injury in measles virus infection. III. Presence and characterization of human cytotoxic lymphocytes

Human peripheral blood leukocytes (PBL) from patients with chronic measles virus infection (SSPE) or from immune, adult humans (convalescent from acute childhood measles virus infection) are cytotoxic for target cells infected with measles virus as measured by a 51Cr assay. Specific release of 51Cr by immune PBL occurred both with and without human antibodies added to measles virus-infected cultures. Maximal killing in the absence of added antibodies to measles virus was usually detected only after 15 to 18 hr of incubation and with a high PBL to target ratio (100:1). When antibody to measles virus was added, PBL-mediated killing of virus-infected cells was not blocked. Instead, killing was enhanced and maximal lysis occurred with fewer PBL and a shorter incubation time. This cytotoxic reaction was inhibited in a dose-response manner upon the addition of Fab fragments of IgG containing antibodies to measles virus. On the average 4 to 5 x 105 antibody molecules bound per infected target cell before initiation of antibody-enhanced PBL killing. Depletion of either glass-adhering or E-rosette-forming cells did not reduce PBL killing of measles virus-infected target cells in either system. In contrast, removal of non-E rosette or of EAC rosette-forming population of PBL almost completely abrogated cytotoxicity. When Fc-bearing cells were removed, killing of virus-infected target cells was concomitantly reduced. Lysis of measles virus-infected target cells did not require histocompatibility between the PBL and the target cell. Further, immunospecific lymphocyte killing was not enhanced by such a histocompatibility fit. These experiments indicate first, that the effector PBL involved in lysis of measles virus-infected targets are not T cells but are probably K cells and, second, that PBL obtained from patients with SSPE are competent in killing measles virus-infected targets. Moreover, sera from SSPE patients did not contain a factor(s) that blocked PBL-mediated cytotoxicity.     

Laboratory diagnosis of subacute and acute encephalitis probably of viral origin: seven years of experience

The results obtained in the laboratory diagnosis of 609 cases of acute or subacute encephalitis, studied during a period of time of even years, is briefly presented. Diagnostic methods included virus isolation from stools and cerebrospinal fluid (CSF); specific serology in serum; detection of intrathecal production of IgG antibody; and, in the last two years, detection of viral genome sequences in CSF by the polymerase chain reaction. Significant results were obtained in 196 cases (32.2%) and the alfa-herpesviruses were responsible for a major part of them (77.5%). Furthermore, 18 cases were likely to respond to dual infection by both herpes simplex and varicella-zoster viruses. Epstein-Barr virus and Human herpesvirus 6 were found in CSF from three immunocompetent patients. Besides the current vaccination program, measles virus is still responsible for an important part (22/196, 11.2%) of cases of viral encephalitis.     

Value of antibody titers for diagnosis of neuroborreliosis

Neuroborreliosis is a very frequent subtype of infection with Borrelia burgdorferi. Because of the widely spread inapparent infections finding of diagnosis by analysis of serum antibodies is very difficult. In the years 1990-1994 the serum of 6.775 patients of the Department of Neurology in Homburg, Germany was analysed with regard to Borrelia burgdorferi specific IgG antibodies. 24% showed a positive serum titer and 20% a borderline result. 73 patients showed a specific intrathecal IgG antibody synthesis. In contrast to patients with antibodies in serum these patients showed a significant cumulation during summer. The high percentage of positive serum titers and the season independence support the assumption of widely spread inapparent infections. If a patient shows neurological symptoms the finding of serum antibodies against Borrelia burgdorferi is not sufficient for the diagnosis of Neuroborreliosis. A specific intrathecal synthesis of antibodies, is the most reliable serological indicator for Neuroborreliosis. Intrathecal synthesis usually starts three to four weeks after the first clinical symptoms.     

Encephalitis in ferrets caused by a nonproductive strain of measles virus (D.R.) isolated from patient with subacute sclerosing panencephalitis

A nonproductive, syncytiogenic strain (D.R.) of measles virus, isolated from a patient with subacute sclerosing panencephalitis (SSPE), was inoculated intracerebrally into ferrets in an attempt to induce subacute encephalitis. Inoculation of freeze-thawed syncytia before immunization was the least effective procedure, and inoculation of live syncytia after immunization with measles virus vaccine was the most effective procedure, for induction of subacute or persistent subclinical encephalitis in the animals. After the latter procedure three of five ferrets developed subacute or subclinical encephalitis, whereas ferrets inoculated with live syncytia without prior immunization consistently contracted acute fatal encephalitis in one to two weeks. The subacute encephalitis in ferrets was characterized by high titers of antibody to measles virus in serum. At the time of sacrifice 1.25, 4.5, or 8.0 months after inoculation, brains of the ferrets showed histologic lesions similar to those characteristic of SSPE, and nonproductive syncytiogenic measles virus was recovered from the brains of two of the animals. All three ferrets had greatly increased concentrations of gamma-globulin in their brains and high levels of neutralizing and hemagglutination-inhibiting antibodies to measles virus. Only one of these animals developed clinical signs 1.25 months after inoculation.     

Surgical management and seizure outcome in patients with tuberous sclerosis

The central nervous system is considered an early and common target for the human immunodeficiency virus type 1 (HIV-1). Serum and cerebrospinal fluid (CSF) from 20 HIV positive patients, including 14 with AIDS-dementia complex (CDC stage IV) and 6 asymptomatic individuals (CDC stage II) were analyzed by enzyme immunoassay for detection of antibodies to native myelin basic protein (MBP) and for the amino acid sequence 68-84 exposed after partial degradation of native MBP. Control groups included HIV-1 negative patients with degenerative and/or vascular dementia, chronic multiple sclerosis (MS) and individuals without any sign of neurological or cognitive disturbances. As opposed to control groups, serum and CSF samples from MS and HIV-1 infected patients showed several oligoclonal bands running in the gamma region. AIDS-dementia complex (ADC) patients had increasingly high intrathecal IgG specific antibody titers for the amino acid sequence 68-84 of MBP. Marked intrathecal antibody production for myelin components was also detected in the majority of HIV-1 infected asymptomatic individuals. Such alteration paralleled development of cognitive deficits, neurological abnormalities and appearance of CNS demyelinating plaques. Preferential immune recognition of this myelin epitope within the CSF during early stages of HIV-1 infection might point to an ongoing process of active demyelination and ultimately indicate subclinical CNS involvement.     

Antibodies to varicella zoster virus in the cerebrospinal fluid of neonates with seizures

Four neonates with convulsions had IgG antibodies in their cerebrospinal fluid (CSF) to varicella zoster virus (VZV). These antibodies were found in the sera of two of these patients after the age of 6 months. Antibodies to 16 different microbes were studied from the serum and CSF of 201 neonates with neurological problems. The presence of DNA specific to HSV-1, HSV-2, and VZV in the CSF was also investigated using the polymerase chain reaction (PCR). Antibodies to VZV were detected in the CSF of four neonates. Antibody indices suggested production of VZV specific antibodies in the central nervous system. These findings suggest that intrathecal production of antibodies to VZV can appear in neonates with neurological problems, which suggests that intrauterine VZV infection can be acquired without cutaneous symptoms in the mother.     

Long-term findings in patients with facial palsy and antibodies against Borrelia burgdorferi

Little is known about the long-term effects of Borrelia burgdorferi (Bb) infection in untreated patients with peripheral facial palsy. We investigated 12 patients with elevated serum Bb antibody levels, with a median follow-up time of 11 years, during which 3 of the 12 still exhibited intrathecal antibody production of antibodies against Bb flagellar antigen, and 2 of the 3 had normal serum Bb antibodies. Four of the 12 had elevated serum antibody titres at the late follow-up examination. Arthralgia, reported by 7 patients, was the single most common complaint. Four patients showed extensive oculomotor disturbances, which were not correlated to antibody titres or intrathecal antibody synthesis. In 1 of the patients with intrathecal Bb antibody production, most symptoms were eradicated by antibiotic treatment 6 years after the initial infection. We conclude that even several years after a Bb infection, intrathecal Bb antibody production can still occur in serum Bb IgG antibody negative patients with a history of facial palsy.     

Effects of dopaminergic drugs on food- and cocaine-maintained responding. IV: Continuous cocaine infusions

We modified and optimized a new microplate hybridization assay to detect the varciella-zoster virus (VZV) PCR product, and studied cerebrospinal fluid (CSF) samples of 287 patients with meningitis, encephalitis or other neurological diseases or symptoms. Specific antibodies to VZV and reference antigens were determined by enzyme immunoassay from serum and CSF, they were then compared with clinical findings and with the results obtained by VZV-PCR using different detection methods for VZV-specific amplified DNA. VZV DNA was found in the CSF of 25 patients using the microplate hybridization assay and chemiluminescence detection for amplified DNA. All 25 CSF samples were also positive in Southern blotting. Among the patients, 10 had chickenpox, 4 had shingles, and 11 had no rash at all. The detection rate of VZV-specific DNA by microplate hybridization was 30% higher than that obtained by conventional agarose gel electrophoresis. In most patients the diagnosis was confirmed by demonstrating specific intrathecal antibody production to VZV but not to other viruses. These results indicate the presence of VZV in the central nervous system (CNS) in many patients with chickenpox or shingles, and even in patients without a rash. The microplate hybridization assay based on chemiluminescence detection improves considerably the detection rate of the VZV-PCR product compared to agarose gel electrophoresis and will add to the list of recognized VZV infections in the CNS. It is especially useful in cases where there is no cutaneous manifestation.     

Enzyme-linked immunosorbent assay for serological survey of equine arteritis virus in racehorses

To examine antibodies against equine arteritis virus (EAV), an enzyme-linked immunosorbent assay (ELISA) using purified virus antigen was developed. The results of ELISA were compared with those of serum neutralization (SN) tests. The ELISA absorbance values and the SN titers in sera collected weekly from EAV-infected horses showed a similar pattern. The ELISA could detect antibody to EAV in horses experimentally infected with not only a homologous virus strain, which was used as the ELISA antigen, but also a heterologous strain. Using the ELISA, serum samples collected in 1996 from racehorses in three prefectures (Hokkaido, Ibaraki, and Shiga) were examined and there was no evidence of recent EAV infection among these racehorse populations in Japan. The ELISA should be a simple and highly specific method for rapid screening of EAV infection in racehorses.     

Antibodies to the axolemma-enriched fraction in the cerebrospinal fluid and serum of patients with multiple sclerosis and other neurological diseases

Antibodies to an axolemma-enriched fraction (AEF) antigen have been detected in the cerebrospinal fluid (CSF) and serum of patients with Multiple Sclerosis (MS) using an enzyme-linked immunosorbent assay (ELISA). A marginal elevation (P < 0.08) of anti-AEF IgG was found in MS CSF when compared with OND samples. When CSF was diluted to a standardized IgG concentration, the anti-AEF IgG level in MS CSF was significantly elevated (P=0.007) when compared to OND CSF. MS serum was also found to contain a significantly higher level (P < 0.001) of anti-AEF IgG when compared to OND serum using the ELISA technique.     

Influence of the specific T cell response on seroconversion after measles vaccination in autologous bone marrow transplant patients

Six patients who were seronegative to measles after autologous bone marrow transplantation (ABMT) were vaccinated with a live attenuated measles vaccine. The specific T helper cell response was studied by measuring lymphocyte proliferation induced by measles antigen and B cell response by measles specific IgG by ELISA. Blood samples were drawn before, at 1-3 months, and at 1 year after vaccination. It was found that a pre-existing T cell response correlated with an impaired B cell response 1 year after vaccination (r = 0.83, P = 0.04), whereas no correlation was found between IgG titers before vaccination and IgG titer increase, or T cell response after vaccination. Furthermore, there was a transient negative correlation between the T cell response at 1-3 months after vaccination and the T cell response before vaccination (r = -0.90, P = 0.04) that became positive at 1 year after vaccination (r = 0.90, P = 0.02). In conclusion, in patients seronegative to measles who were revaccinated with measles vaccine after ABMT, a pre-existing T cell response correlated with an impaired B cell response, while pre-existing low-level IgG antibodies had no significant influence on the IgG titer rise. A sustained T cell response to measles antigen before vaccination may thus be one possible explanation for measles vaccine failure in ABMT patients.     

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus)

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.     

Intrathecal synthesis of immunoglobulins in eosinophilic meningoencephalitis due to Angiostrongylus cantonensis

Eosinophilic meningoencephalitis due to the nematode Angiostrongylus cantonensis, which is endemic to Cuba, occurs in children and is due to accidental contact with soil snails. The course is less often fatal than in adult patients in southeastern Asia. Cerebrospinal fluid (CSF) and serum samples from 24 pediatric patients were analyzed and evaluated in CSF/serum quotient diagrams (Reiber graphs) to characterize the neuroimmunological response and the blood-CSF barrier dysfunction that occur in the course of the disease. At the time of the first diagnostic lumbar puncture, together with eosinophilic pleocytosis (1,920 +/- 400 cells/microl), intermediate blood-CSF barrier dysfunction (i.e., an increased CSF/serum albumin quotient) with no intrathecal immunoglobulin G (IgG), IgA, and IgM class response was observed in all cases. Seven days later, at the time of early clinical recovery, the blood-CSF barrier dysfunction was normalized in 75% of the patients, but meanwhile, intrathecal immunoglobulin synthesis emerged in all cases, as either a two-class response (IgG and IgA in 85% of the patients) or a three-class response (IgG, IgA, and IgM; 30%). The fraction of eosinophilic cells (40%) remained large despite a decreasing total cell count. The neuroimmunological pattern of this inflammatory response to the parasite and its toxins is discussed with regard to the CSF patterns of other infectious diseases caused by bacteria or viruses.     

Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.     

Relationship between gingival crevicular fluid and serum specific antibody titers in periodontitis and changes after the treatment

Gingival crevicular fluid (GCF) samples were collected from 80 teeth in 20 periodontitis patients (13 RPP, 7 AP) before and 1 month, 7 months after treatment. Serum samples were taken at the same time. 12 healthy subjects (48 teeth) were chosen as the control group (H). The levels of IgG antibody to Bacteroides gingivalis was measured by ELISA. The relationship between serum and GCF specific antibodies was assessed. Before treatment, the mean ratio of antibody in GCF and serum (the GCF/Sr ratio) in both RPP and AP group was lower than 1, and significantly lower than that in H group. After treatment, the serum antibody titers greatly reduced while GCF antibody increased at 1 month after treatment and decreased at 7 months after treatment. The GCF/Sr ratio raised to greater than 1 in both RPP and AP. The elevation of GCF antibody may be associated with the lower antibody consumption caused by decreasing amount of B. gingivalis in pocket, and/or associated with the local antibody synthesis. It was suggested that the GCF/Sr ratio of antibody level might be used as a significant indicator in evaluation of treatment effectiveness.     

Regulation of thrombosis and hemostasis by antithrombin

An ELISA has been set up for quantifying mouse monoclonal antibodies in culture supernatant. The assay includes rabbit anti-mouse IgG antibodies chromatographycally purified. This preparation was used as coating and as conjugated antibodies in the ELISA. The assay can detect IgG1 with sensitivity of 0.2 ng/mL, IgG2a (0.85 ng/mL), IgG2b (0.13 ng/mL), and IgG3 (3.19 ng/mL) in culture supernatants. The effective working range was from subnanogram per mL quantities to 30 ng/mL by using a computer statistical program. Variation coefficient of ELISA was below 7%. Correlation estimates with a similar ELISA using commercial reagents were performed for each mouse antibody subclass. The assay was able to detect the four mouse monoclonal antibody subclasses in pure human serum as compared with the same ELISA using commercial antibodies. A 24-h pharmacokinetic profile of 1 patient treated with an IgG2a monoclonal antibody is presented.