A combined indirect elisa and immunoblotting for the detection of intrathecal herpes simplex virus igg antibody synthesis in patients with herpes simplex virus encephalitis

A combined indirect ELISA and immuno-blotting assay was used for the detection of intrathecal synthesis of IgG antibodies to herpes simplex virus (HSV) in patients with HSV encephalitis (HSVE). By using these two assays as well as three markers for blood-brain barrier, leakage can be easily excluded. A total of 21 sera and 24 cerebrospinal fluid (CSF) samples from 11 patients with HSVE were examined. For seven patients more than one pair of serum and CSF were available. For one patient IgG antibodies began to be detectable in CSF after the sixth day from the onset of the disease. In the other 10 patients the intrathecal synthesis of HSV IgG antibodies was detected later than the sixth day and reached high optical density (OD) values after the 10th day from the onset of disease, at the earliest. In contrast, intrathecal HSV antibody synthesis was not found in specimens taken from 20 patients with acute meningitis who composed our negative control group. The use of a combined indirect ELISA and of an immunoblotting assay on a single dilution of serum and CSF for HSV IgG synthesis in the central nervous system (CNS) allowed the diagnosis of HSVE after the first week of disease.     

Serodignosis of herpes simplex encephalitis by antibody capture enzyme-linked immunosorbent assay

The sensitivity of a herpes simplex virus (HSV) IgG capture enzyme-linked immunosorbent assay was tested in 103 cerebrospinal fluid (CSF) and serum samples from 47 patients with herpes simplex encephalitis (HSE). Significant differences between CSF and serum absorbances were found in all HSE patients; earlier in three out of 30 patients by the capture technique than by the indirect ELISA. In follow-up samples with exceedingly high anti-HSV activity, maximal absorbance values were found in CSF and serum resulting in falsely-negative absorbance differences. Reanalysis with a lower concentration of capture anti-IgG solved the problem. This lower anti-IgG concentration was found suboptimal for analysis of early specimens. The specificity was tested in specimens from 115 patients without intrathecal anti-HSV production as determined by indirect ELISA. Five out of 73 patients with focal encephalitis of non-HSV origin had low level positive capture results. Four of them had varicella zoster (VZ) meningoencephalitis. Severe blood-CSF barrier damage did not interfere in seven patients tested. The IgG-capture was found advantageous to the indirect technique as the results were more sensitive and clearcut. It is less labour intensive and may be completed within five to six hours.     

A simple method to detect intrathecal production of specific antimeasles antibodies in cerebrospinal fluid during subacute sclerosing panencephalitis

The intrathecal production of antimeasles antibodies was studied using the enzyme-linked immunosorbent assay in eight specimens of serum and cerebrospinal fluid (CSF) from patients with clinical signs of subacute sclerosing panencephalitis (SSPE). The test was performed using a 1:5 dilution of CSF and a 1:2000 dilution of serum (ratio 1:400) in order to nullify the physiological gradient of immunoglobulins across the blood brain barrier (BBB). This procedure allowed a rapid and accurate assessment of the synthesis of specific immunoglobulins in the CSF and a good evaluation of the permeability of the BBB. A diagnosis of SSPE was provided in five out of eight patients with clinical signs of the disease. Clinical follow-up confirmed the diagnosis of SSPE in the group of patients with clear evidence of intrathecal synthesis of antimeasles antibodies.     

Intrathecally produced antibodies to Borrelia burgdorferi measured by IgG-capture ELISA

An IgG-capture enzyme-linked immunosorbent assay (ELISA) which directly measures the intrathecal production of Borrelia burgdorferi-specific IgG in neuroborreliosis is described. The method is based on the simultaneous measurement of IgG-specific antibodies in paired cerebrospinal fluid (CSF) and serum samples. Sixty-nine paired CSF/serum samples from 64 patients with neuroborreliosis or other neurological disorders and six samples from acrodermatitis chronica atrophicans (ACA) patients were analysed by IgG-capture ELISA. Of 12 samples from patients with early neuroborreliosis, intrathecal production of B. burgdorferi-specific IgG could be demonstrated in 11 samples. Of 56 patients with other neurological disorders of known or unknown etiology, intrathecal production of B. burgdorferi-specific IgG was demonstrated in one patient who had a history compatible with a previous neuroborreliosis. No intrathecal production was found in the samples from ACA patients in spite of high Borrelia titres in their CSF as measured by indirect ELISA. In conclusion, the B. burgdorferi IgG-capture ELISA is a sensitive and reliable method for the detection of intrathecal production of IgG antibodies to B. burgdorferi. It is advantageous in comparison to the indirect ELISA as it does not require correction for leakage across the blood/CSF barrier.     

Simultaneous expression of Borrelia OspA and OspC and IgM response in cerebrospinal fluid in early neurologic Lyme disease.

Lyme disease is the major tick-borne disease, caused by Borrelia burgdorferi (Bb). Neurological involvement is common in all stages. In vivo expression of Bb antigens (Ags) and the immune response to them has not been well investigated in the cerebrospinal fluid (CSF). Upregulation of outer surface protein (Osp) C and concomitant downregulation of OspA before tick inoculation of the spirochete has been reported in skin and blood in animals. CSF OspA Ag in early disease suggests otherwise in CSF. Early Ag expression and IgM response in human CSF was investigated here. Paired CSF and serum was collected from 16 early, predominantly erythema migrans Lyme disease patients with neurologic problems, 13 late Lyme disease patients, and 19 other neurologic disease (OND) controls. Samples were examined for IgM reactivity to recombinant Bb-specific Osps using ELISA and immunoblot. Of 12 early Lyme disease patients with neurologic involvement with both CSF and serum IgM against OspC, 7 (58%) had IgM to OspA (n = 5) or OspB (n = 2) that was restricted to the CSF, not serum. Overall, 12 of 16 (75%) of these early Lyme disease patients with neurologic involvement had CSF and serum IgM against OspC. Only 3 of 13 (23%) late Lyme disease patients and none of 19 OND controls had CSF IgM directed against OspC. In conclusion, in CSF, OspC and OspA can be coexpressed, and IgM response to them occurs in early Lyme disease patients with neurologic involvement. This biologic finding may also provide a discriminating marker for CNS infection in Lyme disease.     

Kappa free light chains in cerebrospinal fluid as markers of intrathecal immunoglobulin synthesis

BACKGROUND: Intrathecal immunoglobulin synthesis is observed in several inflammatory disorders of the central nervous system, but its detection by current laboratory tests is either tedious or relatively insensitive. We assessed the diagnostic accuracy of an assay for kappa free light chains (kappaFLC) in cerebrospinal fluid (CSF) and serum, and compared it with traditional tests for intrathecal immunoglobulin synthesis. METHODS: kappaFLCs were measured by nephelometry in CSF/serum pairs from 112 patients. Samples were excluded if blood contamination of CSF as a result of traumatic lumbar puncture (n = 12) or monoclonal bands in both CSF and serum (n = 5) were present. The remaining sample pairs were grouped according to the presence (n = 71) or absence (n = 24) of oligoclonal bands. Data were analyzed as kappaFLC concentrations in CSF, as kappaFLC CSF/serum ratios, and by use of the quotient diagram described previously for immunoglobulins. RESULTS: Both kappaFLC concentrations in CSF and the kappaFLC CSF/serum ratio identified patients with oligoclonal bands with high specificity and sensitivity. The areas under the ROC curves were 0.991 (95% confidence interval, 0.944-0.998) and 0.978 (0.924-0.996), respectively. Exclusion of patients with impaired blood-CSF barrier function further improved diagnostic accuracy. To account for patients with impaired blood-CSF barrier function, data were also analyzed in a quotient diagram. Only two patients without detectable oligoclonal bands would have been misclassified by this approach. CONCLUSIONS: Our data indicate that the nephelometric assay for kappaFLCs in CSF reliably detects intrathecal immunoglobulin synthesis. This automated and quantitative method could simplify the diagnostic procedure for CSF analysis in the routine laboratory.     

Limitation of serological testing for Lyme borreliosis: evaluation of ELISA and western blot in comparison with PCR and culture methods

The aim of the study was to evaluate a one-step procedure using an ELISA test of high specificity and a two-step procedure using immunoblot as a confirmation test, and to compare the results of serological testing with detection of bacterial DNA and living spirochetes. Sera, synovial (SF) and cerebro-spinal fluids (CSF) were obtained from 90 patients with clinical symptoms of Lyme borreliosis. Serum samples were tested with recombinant ELISA and Western blot assay. Citrated blood, cerebrospinal and synovial fluids samples were cultured in cell line and tested by PCR to detect spirochetes. No correlation was found between levels of specific B. burgdorferi antibodies detected with a recombinant antigen ELISA and the number of protein fractions developed with these antibodies by immunoblot. Moreover, Lyme borreliosis patients who have live spirochetes in body fluids have low or negative levels of borrelial antibodies in their sera. This indicates that an efficient diagnosis of Lyme borreliosis has to be based on a combination of various techniques such as serology, PCR and culture, not solely on serology.     

Intrathecal synthesis of specific immunoglobulin G antibodies in neurocysticercosis: evaluation of antibody concentrations by enzyme-linked immunosorbent assay using a whole cysticercal extract and cyst vesicular fluid as antigens

The demonstration of intrathecal antibody production has proven useful for showing the involvement of the central nervous system (CNS) in several diseases. In the present study, the intrathecal synthesis of cysticercus-specific immunoglobulin G (IgG) antibodies was investigated in 30 patients with neurocysticercosis based on calculation of the specific IgG antibody index (AI(IgG)). An AI(IgG) > or =1.5 was considered to be indicative of intrathecal antibody production. Antibody concentrations in serum and cerebrospinal fluid samples were evaluated using an enzyme-linked immunosorbent assay with 2 antigen preparations from Taenia solium cysticerci, namely, a whole cysticercal extract (TsoW) and the vesicular fluid of the parasite (TsoVF). Intrathecal, cysticercus-specific IgG antibody synthesis was observed in 21 (70%) and 23 (76.6%) patients using the TsoW and TsoVF antigens, respectively. The detection of the intrathecal synthesis of specific antibodies may be a potentially useful tool in establishing the involvement of CNS in cysticercosis.     

Epidemiology and diagnosis of Lyme borreliosis

The multisystem disease Lyme borreliosis is the most frequent tick-transmitted disease in the northern hemisphere. In Europe Lyme borreliosis is most frequent in Central Europe and Scandinavia (up to 155 cases per 100,000 individuals) and is caused by the species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. The recently detected genospecies A14S may also play a role in skin manifestations. Microbiological diagnosis in European patients must consider the heterogeneity of borreliae for development of diagnostic tools. According to guidelines of the USA and Germany, serological diagnosis should follow the principle of a two-step procedure (enzyme-linked immunosorbent assay (ELISA) as first step, if reactive; followed by immunoblot). The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens (p100, p58, p41i, VlsE, OspC, DbpA) including those expressed primarily in vivo (VlsE and DbpA) instead of whole cell lysates. VlsE is the most sensitive antigen for IgG antibody detection, OspC for IgM antibody detection. At present, detection rates for serum antibodies are 20%-50% in stage I, 70%-90% in stage II, and nearly 100% in stage III Lyme disease. Detection of the etiological agent by culture or polymerase chain reaction (PCR) should be confined to specific indications and specialized laboratories. Recommended specimens are skin biopsy specimens, cerebrospinal fluid (CSF) and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50%-70%) and synovial tissue or fluid (50%-70% with PCR). CSF yields positive results in only 10%-30% of patients except when the duration of symptoms is shorter than 2 weeks (50% sensitivity). Methods which are not recommended or adequately documented for diagnosis are antigen tests on body fluids, PCR of urine, and lymphocyte transformation tests.     

Cerebrospinal fluid findings in patients with symptoms suggesting chronic Lyme borreliosis

BACKGROUND: Patients with non-specific long-lasting symptoms such as headache, concentration disturbances, and vertigo and who have positive serum borrelial antibody titres are often assumed to have chronic Lyme borreliosis. Because of the possibility that they may have central nervous system involvement they are frequently treated with courses of i.v. ceftriaxone. We assessed central nervous system involvement by examining cerebrospinal fluid samples in a group of such patients. PATIENTS AND METHODS: Adult patients who qualified for the study had non-specific symptoms suggesting central nervous system involvement for longer than six months (but without overt clinical signs of such involvement) and positive serum borrelial antibody titres and/or erythema migrans prior to the onset of symptoms. Cerebrospinal fluid was examined in all patients. RESULTS: None of the 77 patients included in the study (median duration of symptoms 18 months) had pleocytosis and there was no isolation of Borrelia burgdorferi sensu lato from cerebrospinal fluid. Mildly elevated protein concentration and intrathecal borrelial IgG antibody synthesis were demonstrated in 16 (21%) and 7 (9.1%) patients, respectively. CONCLUSIONS: In patients with non-specific long-lasting symptoms attributed to Lyme borreliosis but with the absence of overt clinical signs suggesting central nervous system involvement, the findings of cerebrospinal fluid examination are usually in the normal range. Routine treatment of such patients with i.v. ceftriaxone is not to be encouraged.     

Epidemiology and diagnosis of Lyme borreliosis

The multisystem disease Lyme borreliosis is the most frequent tick‐transmitted disease in the northern hemisphere. In Europe Lyme borreliosis is most frequent in Central Europe and Scandinavia (up to 155 cases per 100,000 individuals) and is caused by the species, B. burgdorferi sensu stricto, B. afzelii and B. garinii. The recently detected genospecies A14S may also play a role in skin manifestations. Microbiological diagnosis in European patients must consider the heterogeneity of borreliae for development of diagnostic tools. According to guidelines of the USA and Germany, serological diagnosis should follow the principle of a two‐step procedure (enzyme‐linked immunosorbent assay (ELISA) as first step, if reactive; followed by immunoblot). The sensitivity and standardization of immunoblots has been considerably enhanced by use of recombinant antigens (p100, p58, p41i, VlsE, OspC, DbpA) including those expressed primarily in vivo (VlsE and DbpA) instead of whole cell lysates. VlsE is the most sensitive antigen for IgG antibody detection, OspC for IgM antibody detection. At present, detection rates for serum antibodies are 20%–50% in stage I, 70%–90% in stage II, and nearly 100% in stage III Lyme disease. Detection of the etiological agent by culture or polymerase chain reaction (PCR) should be confined to specific indications and specialized laboratories. Recommended specimens are skin biopsy specimens, cerebrospinal fluid (CSF) and synovial fluid. The best results are obtained from skin biopsies with culture or PCR (50%–70%) and synovial tissue or fluid (50%–70% with PCR). CSF yields positive results in only 10%–30% of patients except when the duration of symptoms is shorter than 2 weeks (50% sensitivity). Methods which are not recommended or adequately documented for diagnosis are antigen tests on body fluids, PCR of urine, and lymphocyte transformation tests.     

Low intrathecal immune response of anti-EBNA-1 antibodies and EBV DNA from multiple sclerosis patients

Numerous studies have been carried out to determine whether an Epstein-Barr virus (EBV) infection can be considered a risk factor for multiple sclerosis (MS), following the evidence of an increase in IgG response to nuclear antigen-1 (EBNA-1) in both serum and cerebrospinal fluid (CSF) from MS patients. However, the possible interaction between EBV and MS has still not been well characterized, and the possible pathogenic role is yet to be determined. A case-control study (76 cases and 75 controls) was conducted to investigate anti-EBV antibodies synthesis in serum and CSF through intrathecal specific IgG synthesis to EBNA-1, as well as the presence of EBV DNA in plasma, peripheral blood mononuclear cells, and CSF from MS patients. Intrathecal EBNA-1 specific IgG synthesis was detected in 6.6% MS patients and in 17.3% controls. No EBV DNA was found in plasma or CSF, and our findings showed no evidence of high intrathecal EBNA-1 specific IgG synthesis or of significant EBV DNA in CSF in MS patients.     

A sensitive assay of transthyretin (prealbumin) in human cerebrospinal fluid in nanogram amounts by ELISA

A sensitive ELISA method for determining transthyretin (prealbumin) in human cerebrospinal fluid (CSF) is described. The method utilizes goat antihuman transthyretin antibody (IgG fraction) for capture and peroxidase conjugated antibody for color development. The assay has a linear range of 1-4 ng transthyretin added per well. The within-day and between-day coefficients of variation are 5.1 and 6.1%, respectively. The concentration of transthyretin in CSF (ranging from 5 to 20 mg per L) correlated significantly with the corresponding serum concentrations (range 170-420 mg/l). This suggests that synthesis of transthyretin in the brain and peripheral tissues is under similar biological control in normal subjects. The transthyretin concentrations in CSF did not correlate with total CSF protein concentration or age of the subject.     

神经梅毒患者血清及脑脊液免疫学指标诊断特征分析

目的探讨神经梅毒患者血清及脑脊液免疫学诊断特点。 方法选取2013年6月至2016年7月首都医科大学附属宣武医院收治的35例神经梅毒患者,其中32例患者行血清与脑脊液免疫学指标检测,回顾性分析35例患者的检查结果,应用Fisher精确检验比较血清组与脑脊液组IgA、IgM的差异,应用卡方检验比较血清组与脑脊液组IgG的差异,并对脑脊液寡克隆条带阳性检出率和脑脊液24 h IgG合成率进行分析。 结果32例神经梅毒患者检测血清免疫球蛋白,有53.13%(17/32)的患者IgG升高,6.25%(2/32)的患者IgA升高,0%(0/32)的患者IgM升高;脑脊液中,有84.38%(27/32)的患者IgG升高,100.00%(32/32)的患者IgA升高,90.63%(29/32)的患者IgM升高;血清与脑脊液IgA、IgM升高率比较,差异有统计学意义(P<0.01),IgG升高率比较,差异无统计学意义(χ²＝7.27,P>0.05)。25例神经梅毒患者行CSF寡克隆电泳,IgG寡克隆条带阳性率为100.00%(25/25),96.00%(24/25)患者脑脊液24 h IgG合成率升高。 结论神经梅毒临床表现多样,是易误诊的可治性疾病。血清及脑脊液IgG多表现为升高,脑脊液IgA、IgM多表现为升高而血清IgA、IgM多表现为正常,脑脊液寡克隆条带阳性,脑脊液24 h IgG合成率升高等免疫学特点对神经梅毒诊断有意义。     

Antibodies to Borrelia burgdorferi in European populations

We compared the antibodies to B. burgdorferi in three different populations in order to evaluate the diagnostic reliability of Lyme borreliosis serologic analysis. The subjects included 25 patients with Lyme borreliosis (Group 1); 50 patients with diseases of unknown cause, B. burgdorferi ELISA-positive in serum and without B. burgdorferi infection (Group 2); and 1,251 individuals without Lyme borreliosis (Group 3). All samples were tested for B. burgdorferi B31 and B. afzelii antigens using ELISA. The positive results of the ELISA B. burgdorferi B31 assay were confirmed with Western blot for the same strain. In Group 3, 162 (12.9%) patients were ELISA positive for B. burgdorferi B31, while only 6 (0.6%) patients had IgG ELISA antibodies to B afzelii. Bands in WB were detected in 104 (8.4%) of the Group 3 subjects. The bands found to be most reliable for the identification of strain B. burgdorferi B31 by IgG WB were those representing the 93, 39, 34, and 23-kDa proteins. Our results show that serologic diagnosis of Lyme borreliosis is far from clearly established. To date, the only reliable criteria are clinical ones correlated with laboratory evidence.     

Characterization of antibody response in patients with Borrelia meningitis

Western blot analysis was used to characterize the antibody response of 38 patients with Borrelia meningitis to different strains of Borrelia spirochetes. Eight strains of Borrelia spirochetes were analysed by SDS-PAGE which showed major proteins of 60, 41, 31·5–34 and 22–23 kD. Immunoblots of all sera, and all except one CSF from patients with clinically active disease showed IgG and/or IgM antibodies to at least one Borrelia protein. Antibodies to a 41 kD protein was the first to appear and patients with a longer duration of neurological disease had antibodies to as many as 19 different proteins. Some of the 40 controls showed weak bands, in some cases with the same location as in the Borrelia infected patients. In comparison with an ELISA assay based on a whole cell sonicate as antigen, western blot was more sensitive in detecting an early antibody response, especially in serum. We conclude that Western blot might be used as a complement for immunological diagnosis of Borrelia infection in selected cases with low or border-line ELISA titers. However, a more sensitive/specific ELISA assay might be developed with a single protein as antigen.     

Protis系统在脑脊液蛋白质分析中的临床应用和评价

目的探讨分析软件Protis在脑脊液(CSF)蛋白质分析中的临床实用价值。方法用散射比浊法平行测定32例患者CSF与血清中免疫球蛋白(IgG、IgA、IgM)和白蛋白(A lb)的含量,并利用分析软件Protis进行数据和图形处理。结果正常11例,单纯血脑屏障受损12例,仅有鞘内合成6例,血脑屏障受损伴有鞘内合成3例。结论Protis软件能辅助临床对中枢神经系统疾病作出初步的鉴别诊断结论,其报告方式为临床提供了一种快速、可靠、直观、系统的辅助诊断手段,值得广泛推广。     

Predictive markers for hepatitis C antibody ELISA specificity in Australian blood donors

The hepatitis C antibody reactivity rate in 91,748 blood donors tested using the ORTHO HCV C-100 ELISA system was 0.51%. Specificity of ELISA positive reactions was measured using a recombinant immunoblot assay (RIBA). The aim of this study was to identify markers in ELISA positive donors which were predictive of a RIBA positive result. Samples from 430 ELISA positive donors were tested by the first generation RIBA, RIBA-1, which incorporates two HCV peptides C-100 and 5-1-1. Fifty-five per cent (236) were positive and 19% (83) indeterminate. Multivariate analysis of gender, age, HCV ELISA OD ratio, alanine aminotransferase (ALT) status and hepatitis B core antibody (anti-HBc) status identified age, magnitude of HCV ELISA OD ratio and anti-HBc status as the only independent predictors of a positive RIBA-1 result. The relative odds of being RIBA-1 positive were 4.6-fold (95% CI 1.3-16.4) higher among donors aged 25-34 years compared with donors less than 25 or greater than 44; 6.1-fold (2.1-17.9) higher if the donor was anti-HBc positive and 273.4-fold (30.9-2417) higher if the HCV ELISA OD ratio was greater than 5.98 compared to those with a ratio less than 1.77. Seventy-eight of the 83 RIBA-1 indeterminates were tested on the second generation RIBA, RIBA-2, which includes two additional HCV peptide, C22 and C33c. Thirty-one per cent (24) were positive and 41% (32) were negative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement in the laboratory recognition of lyme borreliosis with the combination of culture and PCR methods.

BACKGROUND: Lyme disease is a multisystem, multistage infection caused by three genospecies of the Borrelia burgdorferi sensu lato species. The diagnosis of Lyme disease is based on a history of tick-bite, physical examination, and serological tests. In the seronegative patients with Lyme borreliosis symptoms, additional testing should be introduced. METHODS: The study group was composed of 240 hospitalized patients presented with various clinical symptoms suggesting Lyme borreliosis: 221 of the patients with neurological abnormalities and 19 with oligoarticular arthritis. Citrated blood and serum samples were collected from the patients for culture and serological examination, respectively. Moreover, 173 cerebrospinal and 6 synovial fluid samples were tested. New oligonucleotide primers based on B. burgdorferi sensu lato 16SrRNA gene sequences were designed for the detection of the bacteria in blood, cerebrospinal, and synovial fluid specimens with PCR. Levels of specific antibodies were measured in serum, cerebrospinal fluid and synovial fluid samples using ELISA and Western blot. B. burgdorferi spirochetes from blood, cerebrospinal fluid, and synovial fluid samples were cultured in cell line. Extracted and purified B. burgdorferi DNA was identified by PCR with new oligonucleotide primers. Then three genospecies were identified by PCR amplification with other primer sets specific for 16S rDNA and/or by the restriction fragment length polymorphism of 23S(rrl)-5S(rrf). RESULTS: Bacterial DNA were found in samples from 32 patients, including 28 patients with neuroborreliosis and 4 with Lyme arthritis. B. burgdorferi-specific IgM and/or IgG serum antibodies were detected in 14 of these patients. Fourteen strains of Borrelia garini, 4 strains of Borrelia afzelii and 1 strain of B. burgdorferi sensu stricto were identified by PCR. Genospecies were not recognized in 13 specimens. CONCLUSIONS: The procedure can be a rapid and sensitive diagnostic method for the detection of etiological agents in clinical materials derived from patients with the clinical symptoms of Lyme borreliosis. It can be utilized for both basic research as well as routine laboratory diagnosis.     

Intra-CSF administered recombinant adenovirus causes an immune response-mediated toxicity

High doses of adenotk were injected into the cerebrospinal fluid of rats and nonhuman primates (Macaca mulatta). Vector administration was followed by ganciclovir administration for 14 days. Despite the absence of clinical symptoms, analysis of the cerebrospinal fluid (CSF) and histopathological examination of the central nervous system (CNS) of the monkeys (3 weeks after vector injection) were consistent with a viral meningitis. Immunohistochemical analysis of the inflammatory infiltrates in the monkeys revealed the presence of T and B lymphocytes, indicating a combined cellular and humoral immune response to the vector. This latter was supported by the finding of intrathecal anti-adenovirus antibody synthesis. Rats receiving high intrathecal adenotk doses showed a transient and dose-dependent clinical toxicity consisting of lethargy, hyperemic eyes and weight loss. Histopathological examination of the meninges showed a shift from polymorphonuclear infiltrates during the first post-injection days to clusters of mononuclear cells after 7 days. Acute toxicity is probably related to the early, innate immune response to the vector. In a separate experiment, high levels of IL-8 and IL-6, were measured during the first 2-3 post-injection days in the CSF of two monkeys which received intrathecal adenoLacZ. Therefore, these cytokines seem to play an important role in initiating the nonspecific immune response. In one monkey which received adenotk, recombinant adenovirus was cultured from serum samples obtained at the 7th post-injection day. At this time-point, no vector could be isolated from CSF samples. Based on these preclinical data, we recommend careful dose finding for clinical studies that aim to treat patients with leptomeningeal metastases.