Ilkka Seppälä

The complement regulator factor H binds to the surface protein OspE of Borrelia burgdorferi.

Spirochete bacteria of the Borrelia burgdorferi sensu lato complex cause Lyme borreliosis. The three pathogenic subspecies Borrelia garinii, Borrelia afzelii, and Borrelia burgdorferi sensu stricto differ in their disease profiles and susceptibility to complement lysis. We investigated whether complement resistance ofBorreliae could be due to acquisition of the main soluble inhibitors of the alternative complement pathway, factor H and the factor H-like protein 1. When exposed to nonimmune EDTA-plasma, the serum-resistant B. afzelii and B. burgdorferisensu stricto strains bound factor H/factor H-like protein 1 to their surfaces. Assays with radiolabeled proteins showed that factor H bound strongly to the B. burgdorferi sensu stricto strain. To identify factor H ligands on the borrelial surface, we analyzed a panel of outer surface proteins of B. burgdorferi sensu stricto with the surface plasmon resonance technique. The outer surface lipoprotein OspE was identified as a specific ligand for factor H. Using recombinant constructs of factor H, the binding site for OspE was localized to the C-terminal short consensus repeat domains 15–20. Specific binding of factor H to B. burgdorferi sensu stricto OspE may help the pathogen to evade complement attack and phagocytosis.

Complement Evasion by Borrelia burgdorferi: Serum-Resistant Strains Promote C3b Inactivation

ABSTRACT The most characteristic features of the Lyme disease pathogens, theBorrelia burgdorferi sensu lato (s.l.) group, are their ability to invade tissues and to circumvent the immune defenses of the host for extended periods of time, despite elevated levels of borrelia-specific antibodies in serum and other body fluids. Our aim in the present study was to determine whether B. burgdorferi is able to interfere with complement (C) at the level of C3 by accelerating C3b inactivation and thus to inhibit the amplification of the C cascade. Strains belonging to different genospecies (Borrelia garinii, B. burgdorferi sensu stricto, and Borrelia afzelii) were compared for their sensitivities to normal human serum and abilities to promote factor I-mediated C3b degradation. B. burgdorferi sensu stricto and B. afzelii strains were found to be serum resistant. When the spirochetes were incubated with radiolabeled C3b, factor I-mediated degradation of C3b was observed in the presence of C-resistant B. afzelii(n = 3) and B. burgdorferi sensu stricto (n = 1) strains but not in the presence of C-sensitive B. garinii (n = 7) strains or control bacteria (Escherichia coli,Staphylococcus aureus, and Enterococcus faecalis). Immunoblotting and radioligand binding analyses showed that the C-resistant strains had the capacity to acquire the C inhibitors factor H and factor H-like protein 1 (FHL-1) from growth medium and human serum. A novel surface protein with an apparent molecular mass of 35 kDa was found to preferentially bind to the N terminus region of factor H. Thus, the serum-resistant B. burgdorferi s.l. strains can circumvent C attack by binding the C inhibitors factor H and FHL-1 to their surfaces and promoting factor I-mediated C3b degradation.

Complement Inhibitor Factor H Binding to Lyme Disease Spirochetes Is Mediated by Inducible Expression of Multiple Plasmid-Encoded Outer Surface Protein E Paralogs

Borrelia burgdorferi spirochetes can circumvent the vertebrate host’s immune system for long periods of time. B. burgdorferi sensu stricto and B. afzelii, but not B. garinii, bind the complement inhibitor factor H to protect themselves against complement-mediated opsonophagocytosis and killing. We found that factor H binding and complement resistance are due to inducible expression of a wide repertoire of outer surface protein E (OspE) lipoproteins variably called OspE, p21, ErpA, and ErpP. Individual Borrelia strains carry multiple plasmid-encoded OspE paralogs. Together the OspE homologs were found to constitute an array of proteins that bind factor H via multiple C-terminal domains that are exposed outwards from the Borrelial surface. Charged residue substitutions in the key binding regions account for variations between OspE family members in the optimal binding pH, temperature, and ionic strength. This may help the spirochetes to adapt into various host environments. Our finding that multiple plasmid-encoded OspE proteins act as virulence factors of Borrelia can provide new tools for the prevention and treatment of borreliosis.

Integrative approaches for large-scale transcriptome-wide association studies

Many genetic variants influence complex traits by modulating gene expression, thus altering the abundance of one or multiple proteins. Here we introduce a powerful strategy that integrates gene expression measurements with summary association statistics from large-scale genome-wide association studies (GWAS) to identify genes whose cis-regulated expression is associated with complex traits. We leverage expression imputation from genetic data to perform a transcriptome-wide association study (TWAS) to identify significant expression-trait associations. We applied our approaches to expression data from blood and adipose tissue measured in ∼3,000 individuals overall. We imputed gene expression into GWAS data from over 900,000 phenotype measurements to identify 69 new genes significantly associated with obesity-related traits (BMI, lipids and height). Many of these genes are associated with relevant phenotypes in the Hybrid Mouse Diversity Panel. Our results showcase the power of integrating genotype, gene expression and phenotype to gain insights into the genetic basis of complex traits.

Recent rubella virus infection indicated by a low avidity of specific IgG

Rubella-specific IgG in acute-phase sera produces a characteristically altered zone termed “soft hemolysis” in the radial hemolysis test. Here, the soft hemolysis was shown to be a product of the purified IgG1 subclass isolated from acute-phase sera. In contrast, ordinary hemolysis was produced by IgG1 isolated from sera of previous rubella immunity, indicating that the subclass composition of IgG was not involved in the mechanism of soft hemolysis. A novel type of solid-phase immunoassay was developed for the avidity of virus-specific IgG. Acute-phase IgG (with soft hemolysis) was dissociated from rubella antigen in an enzyme immunoassay (EIA) test by hydrogen-bond disrupting agents under conditions where IgG of previous immunity (showing ordinary hemolysis) remained mostly bound. These data suggest that the mechanism of soft hemolysis is the avidity of rubella-specific IgG. The new quantitative avidity EIA was tested with sera taken from 169 subjects. Recent infection could be shown from sera taken weeks or months after primary rubella.

The Four Subclasses of IgG Can Be Isolated from Mouse Serum by using Protein A-Sepharose

We confirmed the findings of Ey and colleagues that mouse IgG is absorbed by protein A‐Sepharose at pH 8.0. Also confirmed was their finding that IgG1 mainly elutes from such a column by means of a buffer with pH 6.0 and that the corresponding pH values for IgG2a and IgG2b were 4.5 and 3.5. We made the new finding that the hulk of IgG2a bearing allotypes a or j eluted already at pH 5, in contrast to IgG2a bearing allotype b. Another newfinding was that IgG3 was mainly eluted at pH 4.5 regardless of the allotype. All four subclasses of IgG could thus be physically separated if the allotype was a or J (the only known exception is allotype b). Separation of IgG2a and IgG3 was achieved even when the allotype was b by using a pH gradient for elution. IgG2a came out al a slightly higher pH than IgG3. Mouse IgG antibodies against group A streptococcal polysaccharide belonged mostly to IgG3 and, to a lesser extent, to IgG2a and IgG2b.

The expanding clinical spectrum of ocular lyme borreliosis

OBJECTIVE To delineate the clinical manifestations of ocular Lyme borreliosis, while concentrating on new symptoms and findings and the phase of appearance of ophthalmologic disorders. DESIGN Observational case series. PARTICIPANTS Ten patients with Lyme borreliosis-associated ophthalmologic findings previously reported from the Helsinki University Central Hospital in addition to 10 new cases that have since been diagnosed. INTERVENTION/TESTING: The patients underwent medical and ophthalmologic evaluation. The diagnosis of Lyme borreliosis was based on medical history, clinical ocular and systemic findings, determinations of antibodies to Borrelia burgdorferi by enzyme-linked immunosorbent assay and immunoblot analysis, the detection of DNA of B. burgdorferi by polymerase chain reaction, and exclusion of other infectious and inflammatory causes. MAIN OUTCOME MEASURES Ocular complaints, presenting ophthalmologic findings, and the stage of Lyme borreliosis were recorded. RESULTS Four patients presented with a neuro-ophthalmologic disorder, five had external ocular inflammation, 10 patients had uveitis, and one had branch retinal vein occlusion. One patient developed episcleritis and one patient developed abducens palsy within 2 months of the infection incident. In the remaining 14 patients in whom the time of infection was traced, the ocular manifestations appeared in the late stage of Lyme borreliosis. Two patients with a neuro-ophthalmologic disorder and one with external ocular inflammation experienced severe photophobia, whereas the main reported symptom of the patients with uveitis was decreased visual acuity. Four patients with external ocular disease and one with a neuro-ophthalmologic disorder experienced severe periodic ocular or facial pain. Retinal vasculitis developed in seven patients with uveitis. CONCLUSIONS Lyme borreliosis can cause a variety of ocular manifestations, which develop mainly in the late stage of the disease. Photophobia and severe periodic ocular pain can be characteristic symptoms of Lyme borreliosis. In the differential diagnosis of retinal vasculitis, Lyme borreliosis should be taken into account, especially in endemic areas.

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.

Childhood physical, environmental, and genetic predictors of Adult Hypertension: the Cardiovascular Risk in Young Finns Study

Background— Hypertension is a major modifiable cardiovascular risk factor. The present longitudinal study aimed to examine the best combination of childhood physical and environmental factors to predict adult hypertension and furthermore whether newly identified genetic variants for blood pressure increase the prediction of adult hypertension. Methods and Results— The study cohort included 2625 individuals from the Cardiovascular Risk in Young Finns Study who were followed up for 21 to 27 years since baseline (1980; age, 3–18 years). In addition to dietary factors and biomarkers related to blood pressure, we examined whether a genetic risk score based on 29 newly identified single-nucleotide polymorphisms enhances the prediction of adult hypertension. Hypertension in adulthood was defined as systolic blood pressure ≥130 mm Hg and/or diastolic blood pressure ≥85 mm Hg or medication for the condition. Independent childhood risk factors for adult hypertension included the individuals own blood pressure ( P <0.0001), parental hypertension ( P <0.0001), childhood overweight/obesity ( P =0.005), low parental occupational status ( P =0.003), and high genetic risk score ( P <0.0001). Risk assessment based on childhood overweight/obesity status, parental hypertension, and parental occupational status was superior in predicting hypertension compared with the approach using only data on childhood blood pressure levels (C statistics, 0.718 versus 0.733; P =0.0007). Inclusion of both parental hypertension history and data on novel genetic variants for hypertension further improved the C statistics (0.742; P =0.015). Conclusions— Prediction of adult hypertension was enhanced by taking into account known physical and environmental childhood risk factors, family history of hypertension, and novel genetic variants. A multifactorial approach may be useful in identifying children at high risk for adult hypertension. # Clinical Perspective {#article-title-37}Background— Hypertension is a major modifiable cardiovascular risk factor. The present longitudinal study aimed to examine the best combination of childhood physical and environmental factors to predict adult hypertension and furthermore whether newly identified genetic variants for blood pressure increase the prediction of adult hypertension. Methods and Results— The study cohort included 2625 individuals from the Cardiovascular Risk in Young Finns Study who were followed up for 21 to 27 years since baseline (1980; age, 3–18 years). In addition to dietary factors and biomarkers related to blood pressure, we examined whether a genetic risk score based on 29 newly identified single-nucleotide polymorphisms enhances the prediction of adult hypertension. Hypertension in adulthood was defined as systolic blood pressure ≥130 mm Hg and/or diastolic blood pressure ≥85 mm Hg or medication for the condition. Independent childhood risk factors for adult hypertension included the individuals own blood pressure (P<0.0001), parental hypertension (P<0.0001), childhood overweight/obesity (P=0.005), low parental occupational status (P=0.003), and high genetic risk score (P<0.0001). Risk assessment based on childhood overweight/obesity status, parental hypertension, and parental occupational status was superior in predicting hypertension compared with the approach using only data on childhood blood pressure levels (C statistics, 0.718 versus 0.733; P=0.0007). Inclusion of both parental hypertension history and data on novel genetic variants for hypertension further improved the C statistics (0.742; P=0.015). Conclusions— Prediction of adult hypertension was enhanced by taking into account known physical and environmental childhood risk factors, family history of hypertension, and novel genetic variants. A multifactorial approach may be useful in identifying children at high risk for adult hypertension.

IgG subclass distribution of antibodies after vaccination of adults with pneumococcal conjugate vaccines.

The serum IgG subclass response of adults to Streptococcus pneumoniae (Pnc) capsular polysaccharides (PS) 6B, 14 and 23F was measured for four Pnc vaccines: the 23-valent PS vaccine or PS-protein conjugates with diphtheria toxoid (PncD), tetanus protein (PncT) or CRM197 protein (PncCRM) carriers. A standardized enzyme-linked immunosorbent assay specific for IgG subclasses was employed. This assay uses pneumococcal reference serum, lot 89-SF, to which anti-Pnc PS IgG subclass concentrations have been assigned. Both IgG1 and IgG2 responses were more frequent and higher in the conjugate groups than in the PS group. IgG subclasses in subjects vaccinated with PS displayed similar IgG2 predominant distribution previously observed in both natural and vaccine-induced antibodies. Antibodies induced by PncT, however, had a significantly altered IgG2/IgG1 ratio (P < 0.05), with a higher proportion of IgG1.