An epigenome-wide association study meta-analysis of educational attainment.

The epigenome is associated with biological factors, such as disease status, and environmental factors, such as smoking, alcohol consumption and body mass index. Although there is a widespread perception that environmental influences on the epigenome are pervasive and profound, there has been little evidence to date in humans with respect to environmental factors that are biologically distal. Here we provide evidence on the associations between epigenetic modifications-in our case, CpG methylation-and educational attainment (EA), a biologically distal environmental factor that is arguably among the most important life-shaping experiences for individuals. Specifically, we report the results of an epigenome-wide association study meta-analysis of EA based on data from 27 cohort studies with a total of 10 767 individuals. We find nine CpG probes significantly associated with EA. However, robustness analyses show that all nine probes have previously been found to be associated with smoking. Only two associations remain when we perform a sensitivity analysis in the subset of never-smokers, and these two probes are known to be strongly associated with maternal smoking during pregnancy, and thus their association with EA could be due to correlation between EA and maternal smoking. Moreover, the effect sizes of the associations with EA are far smaller than the known associations with the biologically proximal environmental factors alcohol consumption, body mass index, smoking and maternal smoking during pregnancy. Follow-up analyses that combine the effects of many probes also point to small methylation associations with EA that are highly correlated with the combined effects of smoking. If our findings regarding EA can be generalized to other biologically distal environmental factors, then they cast doubt on the hypothesis that such factors have large effects on the epigenome.

Synergistic Expression of Histone Deacetylase 9 and Matrix Metalloproteinase 12 in M4 Macrophages in Advanced Carotid Plaques.

OBJECTIVE/BACKGROUND: Expression patterns and association with cell specific gene expression signatures of the epigenetic regulator histone deacetylase 9 (HDAC9) and matrix metalloproteinase 12 (MMP12) in human plaque are not known. METHODS: This was a prospective cohort study. Genome wide expression analysis was performed in carotid, femoral, aortic plaques (n = 68) and left internal thoracic (LITA) controls (n = 28) and plaque histological severity assessed. Correlation and hierarchical cluster analysis was utilised. RESULTS: HDAC9 was associated with MMP12 expression in carotid plaques (r = .46, p = .012) and controls (r = -.44, p = .034). HDAC9 and MMP12 clustered with inflammatory macrophage markers but not with smooth muscle cell (SMC) rich markers. In plaques from all arterial sites, MMP12 but not HDAC9 showed positive correlation (p < .05) with M2 and M4 polarized macrophage markers, and negative correlation with SMC rich signatures. In the carotid plaques, all M4 macrophage markers associated with MMP12 and HDAC9. The negative association of MMP12 with SMC rich signatures was pronounced in the carotid plaques. Neither HDAC9 nor MMP12 associated consistently with plaque stabilisation or thrombosis related genes. Immunohistochemistry further supported the association between HDAC9 and MMP12 in atherosclerotic plaques. CONCLUSION: M4 macrophages are a possible source for HDAC9 and MMP12 expression in advanced human plaques.

Genome-wide association study does not reveal major genetic determinants for anti-cytomegalovirus antibody response.

Cytomegalovirus (CMV) causes an infection, which is followed by a lifelong latency. CMV has received much attention in clinical studies, but little is known about the genetic basis of this common infection. To identify genetic polymorphisms associated with the susceptibility to and strength of anti-CMV immunoglobulin G (IgG) response to CMV infection, we conducted a genome-wide association study (GWAS) using an Illumina BeadChip containing 670 000 probes and participants from the Cardiovascular Risk in Young Finns Study, including 1486 anti-CMV IgG seropositive and 648 seronegative individuals. Statistical analyses were performed using logistic (for susceptibility) and linear regression (for strength of antibody response). None of single-nucleotide polymorphisms (SNPs) was found to be associated with susceptibility to CMV infection at the level of genome-wide significance (P<5 × 10(-8)). Also, none of the association signals identified reached genome-wide levels of statistical significance in the study of the strength of the antibody response to CMV although five SNPs in AGBL1 gene region displayed a suggestive association (lowest P-value=1.86 × 10(-6)). The results indicate that there is no strong evidence of major host genetic factors involved in either susceptibility to or the strength of antibody response to human CMV infection.

Activation of the alternative pathway of complement by monoclonal lambda light chains in membranoproliferative glomerulonephritis.

Immunopathological evidence suggests that activation of the alternative pathway of complement (AP) is involved in membranoproliferative glomerulonephritis (MPGN) and in immunoglobulin A nephropathy. In this report we describe an AP dysfunction-associated factor that was isolated from the serum and urine of a patient with hypocomplementemic MPGN. Extensive glomerular deposits of C3, properdin, and of the terminal complement components were observed in the kidney of the patient. In her serum the AP hemolytic activity was virtually absent. When mixed with fresh normal serum, the patient's serum induced a 96% C3 conversion during a 30-min incubation at +37 degrees C. This activity was found to be due to a circulating factor that by immunochemical characterization proved to be a 46-kD monoclonal immunoglobulin lambda light (L) chain dimer (lambda L). Purified lambda L, but not control lambda or kappa L chains from patients with L chain disease, activated the AP in a dose- and ionic strength-dependent manner. Functionally, lambda L was differentiated from C3 nephritic factor (an autoantibody against the AP C3 convertase, C3bBb) by its inability to bind to and stabilize the C3bBb enzyme. Instead, lambda L was observed to interact directly with the AP control factor H. Thus, lambda L represents a novel type of immunoglobulin-related AP-activating factor with the capacity to initiate alternative complement pathway activation in the fluid phase.

Feasibility of commercial interferon-gamma-based methods for the diagnosis of latent Mycobacterium tuberculosis infection in Finland, a country of low incidence and high bacille Calmette-Guérin vaccination coverage.

The performances of the QuantiFERON-TB Gold in Tubes (QFGT), T SPOT-TB (ELISPOT) and the Mantoux test were compared for the diagnosis of latent tuberculosis infection in Finland, a country of low tuberculosis incidence. In Cohort A (16 students), freshly isolated peripheral blood mononuclear cells (PBMCs), and in Cohort B (21 school children), cryopreserved PBMCs, were used for the ELISPOT assay. Cryopreservation of cells in fetal calf serum, but not in serum-free medium, produced false-positive results. Discrepancies between the results of the assays were observed. It was concluded that the accuracy of these ex-vivo methods needs additional evaluation.

Mouse IgG antibodies have subclass associated affinity differences.

Subclasses of IgG were separated from pools of mouse sera by letting immunoglobulins absorb on protein A-Sepharose and by eluting with buffers of decreasing pH. Most donor mice were immunized with a conjugate of a hapten (NIP) and chicken gamma globulin 20 days previously. The results indicate that concentrations of IgG varied from 5.1 to 8.6 mg/ml in the pools of immune sera and was 3.0 mg/ml in one normal serum tested. One half of this was IgG1, ca. 20% of IgG2a and IgG2b each, and 10% IgG3 in the pools of BALB/c sera. IgG2a and IgG3 could not be separated from C57BL sera (due to allotype b), but their combined share of IgG appears to be higher than in BALB/c. Immune sera contained 0.5-1.6 mg/ml of anti-NIP antibodies. Of this 90-98% was IgG1 and the remainder was split between the other subclasses. Up to one half of the protein in the IgG1 fraction was anti-NIP antibody. This surprising finding was confirmed by demonstrating that nearly 50% of the u.v.-light absorption was specifically removed by a NIP-immunosorbent. Subclass-associated affinity-differences were observed. IgG1 anti-NIP had a greater average affinity than IgG2a anti-NIP antibodies. The difference was ca. 1.5-fold when the equilibrium dialysis was focusing on the high-affinity bracket of the total population (concentration of free hapten 16-200 nM). At higher hapten concentrations the trend was the same but the data are fewer. Antibodies in subclasses IgG2b and IgG3 appear to share the lower affinity of IgG2a.

Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain.

Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phenyloxazolone (Ox) were produced in Escherichia coli. The signal sequences of the Fd and L chains were correctly processed, the fragments were secreted into the periplasmic space and released into the culture medium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 micrograms/ml and 0.5 micrograms/ml, respectively. The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a complete L chain and C-terminally degraded fragments of the Fd chain. A deletion up to the interchain disulfide bond in the first constant domain (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncated Fab fragment. The production level of the truncated fragment was comparable to that of the Ox IgG1 Fab and its hapten-binding activity similar to that of the idiotype monoclonal antibody. The temperature stability of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, suggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.

Intrathecal humoral immunologic response in neurologically symptomatic and asymptomatic patients with human immunodeficiency virus infection.

We analyzed the intrathecal humoral immunologic response in 42 human immunodeficiency virus (HIV)-infected patients. Eighteen patients had clinical neurologic abnormalities, while the remaining 24 patients were neurologically symptom-free. Nine of the neurologically symptomatic patients at early infection had slight neurologic dysfunction; in nine other subjects at late infection, the neurologic impairment was moderate or severe. When compared with symptom-free patients, neurologically symptomatic patients had increased intra-blood-brain barrier (BBB) HIV-specific IgG (p less than 0.001) and total IgG synthesis (p less than 0.01) with oligoclonal bands (OCBs) in the CSF and/or serum (11/18 versus 3/24). At early stages of the infection, neurologically symptomatic patients showed increased total intrathecal IgG synthesis (9/9) coincident with OCBs in the CSF and serum (7/9) and slight mononuclear pleocytosis (7/9), but less frequent HIV-specific IgG production within the CNS (6/9). In advanced infection, the number of neurologically symptomatic patients with intrathecal HIV-specific IgG synthesis (8/9) was higher, while the number of those with increased total intra-BBB IgG synthesis (5/9; p less than 0.01), OCBs (4/9), and increased CSF leukocyte count (1/9; p less than 0.001) was lower than at early infection. Our data suggest humoral intra-BBB immunoactivation at early stages of HIV infection followed by declining B cell response within the CNS at advanced infection.

Disturbance of hapten-antibody equilibria by ammonium sulphate solutions. A source of error in antibody affinity determinations.

The affinity of anti-hapten antibody can be conveniently measured by precipitating immune complexes with ammonium sulphate. The method has, however, not proved very reproducible. Here is described one variable difficult to control in the assay: the ammonium sulphate was found to cause dissociation of ligands from hapten (NIP)--antibody complexes. The reason was the volume increase caused by addition of ammonium sulphate. The study suggested that in the calculation of the free hapten concentration the final volume during precipitation should be used. The precipitate should not be washed when hapten binding capacities are measured.

Immunological methods based on antigen-coupled bacteriophages.

Simple chemicals, many proteins, nucleic acids or polysaccharides can be coupled to bacteriophages without completely destroying their infectivity. The coupled phages are then sensitive to inactivation by the relevant antibodies. Thus it is possible to construct phage inactivation assays for antibodies or inactivation inhibition assays for antigens. The advantages of the bacteriophage methods include superior sensitivity and good stability of the reagents. The phage inactivation method detects preferably high-affinity antibodies, but what really matters is a high avidity. Because of the polyvalency IgM antibodies can have a high avidity associated with moderate affinity, and therefore low amounts of IgM class antibody, for instance natural antibody, are often measurable by this test. The chemical conjugation inactivates a proportion of the bacteriophages. If the antigen can be made chemically reactive (self-coupling), approximately 10% of the phage particles escape this inactivation and can serve as indicators of antibody action. When the antigen cannot be made chemically reactive, this inactivation is more complete and the phage assay is less satisfactory.

ELISA measurement of IgG subclass production in culture supernatants using monoclonal antibodies.

Specific and sensitive ELISA to quantitate the human IgG subclasses in cell culture supernatants are described. These assays detect a minimum of 5 ng/ml IgG1, 90 ng/ml IgG2, 8 ng/ml IgG3 and 8 ng/ml IgG4 and can generally measure IgG subclasses in lymphocyte cultures containing a minimum of 200 ng/ml of total IgG. The isotype specificity of these ELISA is demonstrated and each individual ELISA shown to react with a number of paraproteins of the relevant subclass independently of their light chain type or their (major Caucasian) allotype. These assays have been used to determine the IgG subclass response of normal human lymphocytes to pokeweed mitogen in vitro.

Increased occurrence of free immunoglobulin light chains in cerebrospinal fluid and serum in human immunodeficiency virus-1 infection.

The presence of free immunoglobulin light chains (FLCs) in the cerebrospinal fluid (CSF) and sera of patients with human immunodeficiency virus-1 (HIV-1) infection, multiple sclerosis (MS), and neurologically healthy control individuals was investigated by paying special attention to ensure that only truly free light chains would be detected. The FLCs were extracted by specifically binding them to Sepharose-coupled anti-FLC monoclonal antibodies, and thereafter they were electrophoresed and immunoblotted with monoclonal antibodies to both light chain (LC) isotypes. A frequent occurrence of kappa and lambda FLCs was found in both CSF and sera of HIV-1 infected patients. In HIV-1 infection and in MS, the frequency of FLCs of the CSF was equal. In healthy controls, only occasional weak FLCs were observed in either CSF or serum. FLC bands of the CSF from patients with HIV-1 infection tended to be more intensive than those of the appropriately diluted sera. Both intrathecal synthesis of FLCs and their transudation from sera through the impaired blood-brain barrier (BBB) may contribute to this. Increasing severity of general HIV-1 infection was accompanied by an increase of FLC intensity in sera. A qualitative demonstration of FLC in the CSF may be meaningful only in the absence of altered BBB function.

C3c and C3d fragments of human C3 bind myeloma IgG1 and IgG3 proteins.

Eight human myeloma proteins, two of each IgG subclass, were studied for binding to solid-phase C3c and C3d by the ELISA technique. Myeloma IgG1 kappa, IgG1 lambda, IgG3 kappa and IgG3 lambda proteins bound to C3c and C3d, while two IgG2 kappa, and two IgG4 kappa proteins failed to show significant binding affinity. The results suggest that like C1q, the stable binding sites of C3, located on the C3c and C3d parts of the molecule, have affinity for IgG subclasses 1 and 3.

Establishment of a human lymphoblastoid cell line with specific antibody production against group A streptococcal carbohydrate.

A human lymphoblastoid cell line, secreting specific antibody against Group A carbohydrate (A-CHO) was established by pre-selection of antigen binding normal human lymphocytes, followed by Epsetin Barr virus (EBV) induced immortalization. Culture supernatants were assayed for anti A-CHO antibodies by radioimmunoassay, N-acetyl-glucosamine-coupled T4-phage plaque inhibition tests and passive hemagglutination. As a rule, the supernatants contained about 10 micrograms/ml anti-A-CHO antibodies of the IgM-kappa type. The antibody was fractionated and partially purified on an N-acetyl glucosamine Sepharose 4B column with a recovery of about 3 micrograms/ml of supernatant.

Diagnosis of Lyme borreliosis: non-specific serological reactions with Borrelia burgdorferi sonicate antigen caused by IgG2 antibodies.

ELISA methods that measure IgG class antibodies to sonicated Borrelia burgdorferi may give false positive results. These errors could be traced to non-specific reactivity in subclass IgG2 in several instances. Sera were sampled randomly from two adult populations, which differed in having a high and low incidence of Lyme disease. If the binding of IgG2 subclass antibodies was left unrecorded in the test by the use of monoclonal reagent antibodies selective for IgG1 and IgG3, the frequency of positivity in the ELISA test decreased in samples from the low risk group. Twenty-one samples were found to be positive in an immunoblot confirmatory test. Correct prediction of positivity was obtained for 15 sera by ELISA restricted to IgG1 plus IgG3, for only four sera by ELISA restricted to IgG2 and for only six sera by IgG subclass non-restricted ELISA. A non-restricted ELISA with purified flagella of B. burgdorferi as the antigen predicted correctly 14 of the immunoblot-positive sera. The results of this ELISA correlated well with those of the IgG1 plus IgG3 subclass restricted ELISA in the high risk population (r = 0.95, prevalence of seropositivity 12%), but was significantly worse for the low risk group (r = 0.47, prevalence 2.9%). IgG subclass restriction also decreased cross-reactions of syphilitic sera in the ELISA with sonicated antigen.

Diagnosis and clinical characteristics of ocular Lyme borreliosis.

PURPOSE: To establish a diagnosis, in a group of patients we studied the characteristics of ocular Lyme borreliosis. METHODS: During a two-year period, 236 patients with prolonged external ocular inflammation, uveitis, retinitis, optic neuritis, or unexplained neuro-ophthalmic symptoms were examined for Lyme borreliosis. Antibodies to Borrelia burgdorferi were measured by indirect ELISA and western blot. Cerebrospinal fluid was also analyzed by polymerase chain reaction. RESULTS: Ocular Lyme borreliosis was diagnosed in ten patients on the basis of medical history, clinical findings, and serologic test results. Results of ELISA disclosed that five patients were seropositive, two patients showed borderline reactivity, and three patients were seronegative. Four of the five patients with borderline or negative results by ELISA had a positive result by western blot analysis. In one seropositive patient, polymerase chain reaction verified a gene of B. burgdorferi endoflagellin from the vitreous and cerebrospinal fluid specimen. In five of the six patients with known onset of the Borrelia infection, the ocular disorder appeared as a late manifestation. Abnormalities of the posterior segment of the eye, such as vitreitis, retinal vasculitis, neuroretinitis, choroiditis, and optic neuropathy were seen in six patients. Bilateral paralytic mydriasis, interstitial keratitis, episcleritis, and anterior uveitis were seen in one patient each. CONCLUSIONS: Late-phase ocular Lyme borreliosis is probably underdiagnosed because of weak seropositivity or seronegativity in ELISA assays. Ocular borrelial manifestations show characteristics resembling those seen in syphilis.

HIV-1 specificity of cerebrospinal fluid and serum IgG, IgM, and IgG1-G4 antibodies in relation to clinical disease.

The reactivities of intrathecal and serum IgG and IgM, and IgG1-4 subclass antibodies to various HIV-1 proteins were assessed by immunoblotting at various stages of HIV-1 infection. All patients were examined neurologically including CT and/or MRI, and with HIV-1-specific and nonspecific tests of the cerebrospinal fluid (CSF). In early infection, the occurrence of anti-gag antibodies in both CSF and serum was higher than that of anti-pol antibodies among all IgG subclasses (P < 0.05). Also in late infection, anti-gag IgG1 response was most frequent (P < 0.04), while anti-gag IgG3 and IgG4 reactivities predominated over similar anti-pol antibodies (P < 0.05, respectively). Of anti-pol reactivities, in the CSF of subjects at early infection anti-p32 IgG and IgG1 antibodies were more frequent than in patients at late stages (P < 0.015). In late infection, however, the occurrence of anti-p64 IgM and IgG2-4 antibodies of both CSF and serum was higher than at early stages (P = 0.014). Regarding anti-env response, in patients with advanced infection, the CSF and serum IgG subclass reactivity against gp120 was restricted to IgG1. The CSF of individual patients with HIV encephalopathy showed a higher or similar occurrence of polyisotypic anti-gag and anti-pol IgG3 antibodies than corresponding serum. These results indicate association between declining frequency of anti-pol p32 and anti-env gp120 antibodies and severity of HIV-1 disease.(ABSTRACT TRUNCATED AT 250 WORDS)

Lyme borreliosis in Finland in 1986-1988.

In 1986-1988 there were 123 patients with positive serology for Lyme borreliosis out of 4000 sera referred to the Department of Bacteriology and Immunology, University of Helsinki. Of the 63 patients with positive serology in 1986-1987 20 showed a predominant involvement of the nervous system, 18 complained of joint symptoms and 11 patients merely showed a skin involvement including 8 patients with erythema chronicum migrans (ECM) and 3 patients with acrodermatitis chronica atrophicans (ACA). Two of the patients had unspecific general symptoms and in 5 patients the type of involvement remained unknown. The serology was considered to be falsely positive in 2 patients with tuberculous meningitis, in one with syphilis and in another with recurrent fever.

Treatment of late Lyme borreliosis.

The aim of this study was to develop a treatment for late Lyme borreliosis and to compare the clinical results with serological findings before and after treatment. It was done in the Aland Islands (population 25,000), a region endemic for Lyme borreliosis. The patients were the first consecutive 100 patients from the Aland Islands with late Lyme borreliosis. They were followed for at least 1 year after treatment. The clinical results of treatment were compared with results of analyses of flagellar IgG antibodies to Borrelia burgdorferi done at the time of diagnosis before treatment and up to 12 months afterwards. Short periods of treatment were not generally effective. The outcome was successful in four of 13 treatments with 14 days of intravenous ceftriaxone alone, in 50 of 56 assessable treatments with ceftriaxone followed by 100 days of amoxycillin plus probenecid, and in 19 of 23 completed treatments with ceftriaxone followed by 100 days of cephadroxil. Titres of IgG antibodies to B. burgdorferi flagella declined significantly after 6 and 12 months in the patients who had successful treatments. All patients whose final titres were less than 30% of the initial titre were in the successful group. Their titres usually remained above the upper limit of normal for a long time but a decline to a value of less than 30% of that before treatment was always a sign of cure.

Concentrations of serum immunoglobulins and antibodies to pneumococcal capsular polysaccharides in patients with recurrent or chronic sinusitis.

A study was carried out to search for underlying immunoglobulin deficiencies in 25 patients with recurrent or chronic sinusitis. The mean duration of the patient histories of recurrent or chronic sinusitis was 7.2 years. Concentrations of serum immunoglobulins and specific pneumococcal antibodies were measured in the patients and in 25 age- and sex-matched control individuals. The mean serum IgA concentration (1.6 g/L) was lower in the patients than in the control individuals (2.1 g/L, p = .024). On the other hand, the mean serum concentration of IgG antibodies to pneumococcal type 14 polysaccharide was higher in the patients (2.54 microg/mL) than in the control individuals (0.92 microg/mL, p = .008). However, elevated concentrations of IgG antibodies to pneumococcal type 14 polysaccharide were detected mainly in patients with the highest serum IgA concentrations. The results suggest that in a subpopulation of patients with a long-lasting history of sinusitis, a low serum IgA concentration may be associated with a susceptibility to sinusitis.