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.

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.

Complement evasion by Borrelia burgdorferi: serum-resistant strains promote C3b inactivation.

The most characteristic features of the Lyme disease pathogens, the Borrelia 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.

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 of Borreliae 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. burgdorferi sensu 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.

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.

Allotype-associated differences in concentrations of human IgA2.

Concentrations of immunoglobulin (Ig)A2 were determined in 176 Finnish blood donor sera. Their IgA, IgM, IgE, IgG1, IgG2, IgG3 and IgG4 concentrations and Gm allotypes had been determined earlier. The mean concentration of IgA2 was higher in individuals carrying the Gm(ax) allele (0.15 g/l) than in those negative for Gm(x) (0.103 g/l). The difference was statistically significant (P = 0.006). As > or = 70% of IgA was usually IgA1, its concentration could be calculated fairly reliably by subtracting the IgA2 value from the IgA value. The mean IgA1 concentration (2.03 g/l) seemed to be independent of the Gm allotypes.

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.

Low concentrations of Gm allotypic subsets G3 mg and G1 mf in homozygotes and heterozygotes.

Serum concentrations of IgG3, IgG1, and of the Gm allotypic subsets of these two isotypes were measured in adult homozygotes and heterozygotes. Alleles G3 mb and G3 mst of the IgG3 locus, and alleles G1 ma and G1 max of the IgG1 locus were found to associate with a high concentration of the allotypic product. Alleles G3 mg (IgG3) and G1 mf (IgG1) were associated with a low concentration of the product. This was true regardless of the haplotype; for example, allele G3 mb was associated with a high concentration of the product in all haplotypes f;n+;b f;n-;b and fa;n+;b. One dose of allele G3 mg was associated with a characteristic mean concentration of the product (g-type IgG3). This rule was valid regardless of the other allele of the subject, thus, heterozygotes and G3 mg/g homozygotes had mean concentrations of 0.10 and 0.20 g/liter, respectively, of g-type IgG3. Products of the IgG1 alleles were also simply additive: one dose of allele G1 ma(x) or G1 mf was associated with mean concentrations of 3.63 and 2.84 g/liter, respectively, and two doses with twice these amounts. Only allele G3 mb did not completely follow this rule. We also studied the serum concentrations and the allotype distribution of 41 IgG1 and 31 IgG3 myeloma proteins. The results suggested that the allotype-associated differences in serum concentrations are caused by different numbers of B cells producing allotypic subsets of IgG1 or IgG3, not by different rates of synthesis per B cell.

Proportion of protein A bindable molecules in human IgM and IgA antibodies to seven antigens.

Human IgM or IgA antibodies to seven antigens (tetanus and diphtheria toxoids, and five bacterial polysaccharides) were studied by determining what proportion of these antibodies were bound by staphylococcal protein A. This alternative binding is a marker of VH genes of family 3. Each response was studied in an average of nine individuals. The binding proportion of antibodies to the two toxoids resembled that of total serum immunoglobulins; 13-14% of IgA and 40% of IgM antibodies were bound by protein A. All anti-polysaccharide antibodies had higher proportions of protein A bindable molecules than serum IgM or IgA indicating a bias for VH genes. This excess was high in antibodies to Haemophilus influenzae type b (Hib) and pneumococcal type 14 polysaccharides (> two-fold). It was moderate but statistically significant in antibodies to pneumococcal types 18C and 3 capsular polysaccharides and to C polysaccharide. All vaccinated Finns exhibited the VH3-preference in antibodies to Hib and type 14 polysaccharide.

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.

Antibody responses to hapten in thymectomized mice: extraordinarily pronounced deficiency in IgG1 production.

The effect of thymectomy on the production of antibodies was studied by immunizing mice with hapten-carrier conjugates. Antibody responses were analysed with monoclonal antibody-based quantitative isotype-resolving assays. In spite of bone marrow reconstitution, irradiation without thymectomy caused a long-lasting relative deficiency in responsiveness to T-independent antigens. Even when no visible remnants of the thymus could be observed at the autopsy of thymectomized mice, there appeared to be a gradual recovery of antibody-forming capacity within 4 months, as assessed by the response to a T-dependent antigen. Therefore, some of the thymectomized mice had to be regarded as having recovered with respect to the helper T-cell effect. The antibody responses to T-dependent antigens were improved in all isotypes by a functional T-cell system, but the IgG isotypes seemed to benefit more than IgM. The most conspicuous deficit in antibody production in non-recovered thymectomized mice was observed in the T-dependent responses of the IgG1 isotype (2000-fold reduction in contrast to about 50- to 100-fold in IgG2a, IgG2b, and IgG3).

Differential induction of membrane-associated interleukin 1 (IL-1) expression and IL-1 alpha and IL-1 beta secretion by lipopolysaccharide and silica in human monocytes.

Bacterial lipopolysaccharide (LPS) and silica dust are known to be effective inducers of interleukin 1 (IL-1) in human cultured monocytes. The data reported here show that although the levels of secreted IL-1 were equally high after in vitro stimulation with an optimal dose of LPS or silica, there were two clear differences: (i) the levels of membrane-associated IL-1 (as detected by the comitogenic effect of paraformaldehyde (PFA)-fixed cells or purified membrane fragments on murine thymocytes) were ca. five times higher after LPS stimulation than after silica stimulation, (ii) the secreted IL-1 after LPS stimulation was mainly of the pI 7 (IL-1 beta) type, while after silica stimulation there were equally high amounts of pI 7 and pI 5 (i.e. IL-1 alpha) forms. In both cases the IL-1 active molecules belonged to the 15 kDa class. These data show that the nature of the activating agent has a clear influence on the distribution of the biologically active IL-1 molecules. Moreover, the finding that after silica stimulation the amount of membrane-associated IL-1 (which was recently shown to be of the IL-1 alpha type) was low, while IL-1 alpha in the culture fluid was clearly elevated, suggests that the IL-1 alpha not attached to the cell membrane (or released from it) significantly contributes to the secreted IL-1 pool.

Quantitation of immunoglobulin classes and subclasses in anti-Rh (D) antibodies.

The isotype profiles of anti-Rh (D) antibodies were measured from 16 serum samples with a solid-phase radioimmunoassay that employed monoclonal anti-isotype antibodies. These antibodies had been standardized in another solid-phase system with the aid of monoisotypic antibodies (e.g., anti-tetanus toxoid of isotype IgG4), and this permitted calculations of the relative concentrations of different isotypes in each anti-D population. Of the heavy-chain isotypes, only IgG1 and IgG3 were found in anti-Rh (D) antibodies. The share of IgG1 varied from 100% (2 sera) to 0% (1 serum) of detected units, and the mean proportion was 73.8%. The remainder was IgG3 (mean share 26.2%). The proportions of kappa and lambda immunoglobulins varied greatly in individual sera, in three sera only kappa Ig and in one serum only lambda Ig could be detected.

Adjuvant effect of bacterial LPS and/or alum precipitation in responses to polysaccharide and protein antigens.

A conjugate of a hapten (NIP) and a strongly antigenic protein chicken gamma globulin (CGG), when injected in soluble form into mice, induced weak primary responses, as weak as responses induced by conjugates of NIP (or other haptens) to polysaccharides Ficoll or alpha (-1-6) dextran. Mean concentrations of anti-hapten antibodies on day 14 varied within the range of 37-105 micrograms/ml (C57BL mice) or 14-38 micrograms/ml (CBA mice). The NIP-protein conjugate administered in alum-precipitated form induced 100 times higher primary antibody responses. Alum-precipitation of NIP-Ficoll made it a modestly stronger antigen than soluble NIP-Ficoll. When lipopolysaccharide (LPS) was injected together with any of the soluble antigens, the mice produced plenty of anti-hapten antibodies regardless of whether the antigen was hapten-polysaccharide or hapten-protein conjugate. Concentrations on day 14 varied from around 400 micrograms/ml to approximately 1600 micrograms/ml. LPS had a similar adjuvant effect on antidextran responses. LPS alone induced a polyclonal immunoglobulin production, and the immunoglobulin produced included 'anti-NIP' or 'anti-dextran' detectable in the solid phase antibody assay. These 'antibodies' induced by LPS alone were almost totally mercapto-ethanol-sensitive and poorly detectable by Farr assay or the bacteriophage assay. The response to the LPS+antigen combination was specific for the antigen and included both mercapto-ethanol-sensitive and resistant antibodies.

The percentages of six immunoglobulin isotypes in human antibodies to tetanus toxoid: standardization of isotype-specific second antibodies in solid-phase assay.

The solid-phase radioimmunoassay for human antibodies was improved so that it gave the total concentration as well as the concentrations of IgM, IgA, IgG1, IgG2, IgG3 and IgG4 antibodies, all seven in the same "L units"/ml. This was accomplished by standardization of monoclonal antibodies with monoisotypic antibodies rather than myelomas; myelomas were found unsatisfactory for this purpose. For the first time it was possible to determine the isotype composition of an antibody population in percent terms. The weight equivalent of the L-unit in one hyperimmune tetanus antitoxin preparation was approximately 7.4 ng. The new solid-phase radioimmunoassay was applied to tetanus toxoid antibodies of the booster response. Total concentrations varied from 1700-26 000 L units/ml (13-200 micrograms/ml). Concentrations of IgG1 antibodies were 1600-25 000 units/ml (average 91% of the total) and concentrations of IgG4 antibodies 12-6900 units/ml (average 6.9% of the total). Antibodies of the other four isotypes were not detected in all sera and together they never exceeded 3% of the total.

Crossing-over frequency in the Igh region of the mouse genome.

An analysis of recombinations between strains C57BL and BALB/c at the Igh locus suggest that only strains B X D20 and B X D27 recombined in this region. This implies that the maps of Igh markers in strains C57BL, DBA/2 and BALB/c resemble one another.

LPS greatly enhances the antibody response to hapten-polysaccharide conjugates, but not to hapten-protein conjugates.

In confirmation of earlier findings, we observed that an injection of bacterial lipopolysaccharide (LPS) into mice caused a considerable increase in the serum concentrations of IgM and IgG (total Ig rose three- to four-fold in 7 days), and a corresponding increase in the concentrations of "natural" anti-(3-iodo-4-hydroxy-5-nitrophenyl) acetyl (NIP) and anti-trinitrophenol (TNP) antibodies. Our main purpose was to determine what effect LPS had on antigen-dependent responses. Hapten conjugates of a polysaccharide and of proteins were used as antigens. Hapten-protein conjugates induced a strong anti-hapten antibody response (up to 1 mg/ml of anti-hapten antibodies on day 7). Hapten-polysaccharide conjugates induced only a meagre increase in anti-hapten antibodies from the pre-immunization level (maximal concentration 65 micrograms/ml on day 7). LPS, when injected with the antigen, greatly enhanced the antibody response to the hapten-polysaccharide conjugates (up to 2.6 mg/ml of anti-hapten antibodies on day 7). It had little effect on antibody responses to hapten-protein conjugates. The combination treatment had the same effect on immunoglobulin concentrations as LPS alone.