[Allergy and asthma in Icelandic children - an epidemiological study.].

OBJECTIVE: The prevalence of allergy and asthma is increasing in Western industrialized countries. The etiology of allergy is multifactorial and only partly understood. In an effort to gather information about asthma and allergy in the pediatric population in Iceland, we have evaluated on a regular basis a cohort of randomly selected children born in 1987. MATERIAL AND METHODS: The first part of the study included 179 children at the age of 18-23 months (mean age 20 months). Of these, 161 children were re-evaluated at four years of age and 134 at eight years. The evaluation included a standardized questionnaire, clinical examination and skin-prick tests. Asthma, eczema, allergic rhinoconjunctivitis and food allergy were diagnosed according to established criteria. RESULTS: At 20 months of age 42% of the children were diagnosed with asthma or allergic disorders, 45% at four years and 34% at the age of eight years. Initially asthma and eczema were most common, but the prevalence and severity of eczema had decreased at four years of age and the prevalence of asthma decreased between four and eight years. No child was diagnosed with allergic rhinoconjunctivitis before two years of age but 7% of four year olds and 10% at the age of eight years. A quarter of the children had at some stage symptoms compatible with more than one allergic disorder. Two-thirds of the children who were diagnosed with eczema and/or asthma before two years of age, were symptom free at eight years. Thirty-eight percent of eight year old children with allergic symptoms had positive skin-prick tests to the allergens used, most commonly to cats. Seventy three percent of eight year old children with allergy and/or asthma, had a first degree relative with a history of allergies. CONCLUSIONS: As in other Western industrialized societies asthma and allergic disorders are common health problems amongst children in Iceland. However, the majority of children with allergic manifestations during the first two years of life, became symptom free before the age of eight years. Conversely, 50% of eight year olds with asthma or allergies were symptom free during the first two years of their life. This suggests that the mechanisms causing allergic symptoms may not be uniform in different age groups.

Enhanced B cell survival in familial macroglobulinaemia is associated with increased expression of Bcl-2.

A family with three cases of macroglobulinaemia of undetermined significance (MGUS), and one case each of immunoblastic lymphoma, Waldentröm's macroglobulinaemia and multiple myeloma was first described 20 years ago. We have previously identified 10 out of 35 healthy family members tested whose lymphocytes produced abnormally high amounts of immunoglobulins in culture. In the present study lymphocyte subpopulations of these hyper-responders have been further characterized and lymphocyte reactivity and survival in vitro have been studied. No differences were detected in the proportions of resting B lymphocytes (CD19+) co-expressing CD5, CD10, CD11b, or CD38, and the CD4/CD8 ratio of T cells was normal before and after stimulation with pokeweed mitogen (PWM). The initial rate of response in terms of immunoglobulin production was not increased, but immunoglobulin levels continued to rise during the second week of culture whereas the production peaked at 8 days in control cultures. This was associated with significantly greater survival of lymphocytes and at 14 days surviving B cells could only be identified in samples from hyper-responders. A lymph node removed because of tuberculosis from a family member 23 years before the diagnosis of multiple myeloma showed very marked Bcl-2 expression in a B cell follicle. This was not seen in a tuberculous lymph node from an unrelated subject. Stimulated cultures from three hyper-responders tested demonstrated significantly higher retention of Bcl-2 in B cells compared with one family control and six unrelated controls. We conclude that the increased production of immunoglobulins previously observed in this family with an inherited tendency for benign and malignant B cell proliferation is the result of enhanced B cell survival, which is associated with increased expression of Bcl-2 following stimulation.

Enzyme immunoassays for measuring complement-dependent prevention of immune precipitation (PIP) and solubilization of preformed antigen-antibody complexes (SOL).

We have developed simple and sensitive enzyme-based methods for evaluating the ability of serum complement to prevent immune complex precipitation (PIP) or to solubilize preformed immune complexes (SOL). Alkaline phosphatase, serving both as antigen and label, is added to goat IgG anti-alkaline phosphatase antibodies, with serum present throughout the assay (PIP), or added after immune complex formation (SOL). After incubation at 37 degrees C for 1 h followed by centrifugation, the enzyme activity of the supernatant, reflecting the amount of immune complexes in solution, is measured by colorimetry. Results are expressed with reference to a standard serum pool assigned 100 arbitrary units (AU). Intra- and inter-assay variabilities are within 10%. The normal ranges were 67-133 AU for PIP and 72-129 AU for SOL. These methods have been standardized for clinical use in relation to impaired complement function and immune complex disease, and adapted for measuring complement mediated binding of immune complexes to erythrocytes. They are sensitive, easy to perform and do not require expensive facilities. By measuring the interaction of complement with immune complexes, these methods may highlight aspects of the classical and the alternative pathway that are different from those detected using haemolysis as an endpoint.

An enzyme based assay for the measurement of complement mediated binding of immune complexes to red blood cells.

A new in vitro method is presented for measuring directly the ability of sera to induce binding of immune complexes (ICs) to erythrocytes (ICRB assay). The assay measures the binding of alkaline phosphatase (AP)-anti-alkaline phosphatase (anti-AP) complexes formed in the presence of the test sera to the complement receptor 1 (CR1) on normal human red blood cells (RBCs). By using a standard serum source, the assay can also be used to measure the IC binding ability of RBCs from different donors. As compared to the traditional CH50 method, the ICRB assay generally showed more pronounced abnormality in 10 individuals tested, of whom 5 had primary deficiency of classical pathway components. Seven out of ten individuals had systemic lupus erythematosus (SLE) and 2/10 had other rheumatic diseases without primary complement deficiency. The ICRB measured in samples from 9 other patients with SLE was significantly decreased when compared to values from 80 normal individuals. ICRB in serum samples from a C2 deficient SLE patient collected during plasma infusion treatment reflected closely the rising amount of C2 in the serum. Using RBCs from different donors ICRB activity correlated well with the numbers of CR1 as measured by a flow cytometric assay (FCA). These methods should be valuable for measuring the overall IC clearance capacity of the blood and have the advantage that the use of radioactive isotopes is avoided.

Characteristics and dynamics of bacterial populations during postantibiotic effect determined by flow cytometry.

Changes in bacterial ultrastructure after antibiotic exposure and during the postantibiotic effect (PAE) have been demonstrated by electron microscopy (EM). However, EM is qualitative and subject to individual interpretation. In contrast, flow cytometry gives qualitative and quantitative information. The sizes and nucleic acid contents of Escherichia coli and Pseudomonas aeruginosa were studied during antimicrobial exposure as well as during the PAE period by staining the organisms with propidium iodide and analyzing them with flow cytometry and fluorescence microscopy. The effects of ampicillin, ceftriaxone, ciprofloxacin, gentamicin, and rifampin were studied for E. coli, whereas for P. aeruginosa imipenem and ciprofloxacin were investigated. After exposure of E. coli to ampicillin, ceftriaxone, and ciprofloxacin, filamentous organisms were observed by fluorescence microscopy. These changes in morphology were reflected by increased forward light scatter (FSC) and nucleic acid content as measured by flow cytometry. For the beta-lactams the extent of filamentation increased in a dose-dependent manner after drug removal, resulting in formation of distinct subpopulations of bacteria. These changes peaked at 20 to 35 min, and bacteria returned to normal after 90 min after drug removal. In contrast, the subpopulations induced by ciprofloxacin did not return to normal until > 180 min after the end of the classically defined PAE. Rifampin resulted in formation of small organisms with low FSC, whereas no distinctive characteristics were noted after gentamicin exposure. For P. aeruginosa an identifiable subpopulation of large globoid cells and increased nucleic acid content was detected after exposure to imipenem. These changes persisted past the PAE, as defined by viability counting. Swollen organisms with increased FSC were detected after ciprofloxacin exposure, even persisting during bacterial growth. In summary, for beta-lactam antibiotics and ciprofloxacin, the PAE is characterized by dynamic formation of enlarged cell populations of increased nucleic acid content, whereas rifampin induces a decrease in size and nucleic acid content in the organisms. Flow cytometry is an ideal method for future studies of bacterial phenotypic characteristics during the PAE.

IgA rheumatoid factor correlates with changes in B and T lymphocyte subsets and disease manifestations in rheumatoid arthritis.

OBJECTIVE: To analyze the relationship between lymphocyte subsets, different rheumatoid factor (RF) isotypes, and clinical features in patients with rheumatoid arthritis (RA). METHODS: Patients with established RA (n = 95) were examined clinically and blood samples were collected for measurements of RF by ELISA and for analysis of lymphocyte subsets by flow cytometry. RESULTS: IgA RF positive patients had more severe disease and higher prevalence of extraarticular manifestations than the other patients. Patients with elevated IgA RF had a higher percentage of CD5+ B cells and of CD4+CD45RO+ T cells compared to the other patients with RA or controls. High percentage of CD4+CD45RO+ T cells was also significantly associated with extraarticular manifestations. Patients with the sicca syndrome had significantly higher ratio of CD5+ B cells than patients without or with other types of extraarticular manifestations. CONCLUSION: Different disease manifestations in RA may be associated not only with certain RF isotypes and RF isotype combinations but also with changes in lymphocyte subsets in the blood. The relative increase of CD4+CD45RO+ T cells in the blood of IgA RF positive patients with RA might reflect preferential recruitment of CD8+CD45RO+ T cells to inflammatory sites.

Detection of orbital implant infection with technetium 99m-labeled leukocytes.

Orbital implant infection is a rare cause of anophthalmic socket pain. Because of the potential danger of infection spreading to nearby structures, it is of paramount importance to diagnose the condition as soon as possible. Scintigraphy is a method for diagnosis of graft infections by radioisotopic imaging of inflammatory sites. We report on a patient with socket pain 3 months after implantation of an acrylic implant. The socket appearance was normal and there were no signs of infection other than culture-positive socket exudation. Three consecutive computed tomography scans revealed no abnormalities. 99mTc leukocyte scintigraphy revealed white blood cell accumulation at the implantation site. The implant was removed and cultured. This produced Staphylococcus epidermidis and R. equii. A parenteral antibiotic treatment was instituted with subsequent improvement of symptoms. Four months later, after negative scintigraphy, a hydroxyapatite implant was inserted, demonstrating full vascularization on a bone scan after 2 months. Two months later, the patient developed the previous symptoms, with all of the former findings, including positive scintigraphy. The implant was removed, revealing a microabscess on the anterior aspect, producing S. epidermidis on culture. We conclude that scintigraphy using 99mTc-labeled leukocytes is a useful technique in diagnosing low-grade orbital infection.

[Cytomegalovirus infections in healthy adults.].

BACKGROUND: Primary cytomegalovirus (CMV) infections in healthy adults are considered extremely rare. To study the extent of this problem in Iceland we undertook a two year (1989-1990) retrospective study of all new CMV infections in adults. METHODS: All positive tests for CMV antibodies in clinical samples (194) were identified in the sole virology laboratory in Iceland. Patients younger than 16 years and all patients with underlying diseases that could cause immunosuppression were excluded (154). The 40 remaining patients were contacted, their case histories reviewed and their serology for CMV, Epstein-Barr and HIV antibodies remeasured. Primary CMV infection was not confirmed in 14 patients leaving 26 immune competent patients who fullfilled our criteria for primary sym-tomatic CMV infection by the presence of IgM anti-CMV antibodies. RESULTS: Duration of illness in the 26 study patients varied from 1 to 25 weeks, usually 7-10 weeks. Fifteen patients were hospitalized. Diagnostic delay was considerable. Immunological tests (DTH skin test, serum immunoglobulines and lymphocyte differential counts) done 172-2 years after the illness did nor reveal any persistent immune abnormalities except for an absolute increase in the number of CD8+ T lymphocytes Conclusions: We conclude that primary CMV infections in adults are not uncommon and probably underdiagnosed. When adult patients present with non-specific symptoms such as low grade fever, malaise and unexplained fatigue, CMV should be considered or excluded with appropriate serological tests.

Cord blood IgE levels are influenced by gestational age but do not predict allergic manifestations in infants.

The predictive value of cord blood IgE (cIgE) for atopy and related disorders was investigated. Samples were collected from 792 infants delivered consecutively at the National University Hospital in Reykjavík in 1987. The concentration of IgE, but not that of IgA, was found to increase with increasing gestational age at birth. There was no correlation between IgE and IgA levels in individual samples. At the age of 18-23 months 180 of these children were studied for manifestations of allergy and related disorders. Included were all available infants with detectable (> or = 0.23 kU/L) cIgE. However, infants born by Cesarean section or with IgA exceeding 10 mg/L were excluded because of potential contamination with maternal blood. The clinical evaluation was made without knowledge of the IgE levels. Sixty-six of the 180 participants (36.6%) were judged to have had definite allergic manifestations. However, no striking correlation was found between allergic symptoms and cIgE levels in this study, nor did high levels of IgE add significantly to the predictive value of family history. Children with atopic features had more frequently been affected by otitis media. Unexpectedly, infants with intermediate cIgE levels (0.2-0.6 kU/L) were significantly less affected by otitis media than children with unmeasurable (< 0.2 kU/L) or high (> or = 0.7 kU/L) cIgE levels. It is concluded that cord blood IgE can not be used to predict allergic manifestations in children under the age of 2 years.

Evaluation of in vivo immune complex formation and complement activation in patients receiving intravenous streptokinase.

The usefulness of several different methods for detecting immune complex formation and complement activation in the circulation were applied to samples from patients receiving intravenous Streptokinase therapy for myocardial infarction. Streptokinase is a foreign antigen and can cause immune reactions. We collected samples from 13 patients, before Streptokinase administration (baseline), at the end of infusion (1 h), 12 h later and on day 7. We measured IgG containing immune complexes (IgG-IC), free C3d and antibodies to Streptokinase by ELISA, and CR1, C3d and C4d on erythrocytes by flow cytometric assay. Antibodies to Streptokinase are common, as all but two of the patients had measurable antibody levels. During Streptokinase treatment there was a drop in antibody levels, most prominent in those patients who had high baseline levels. At the same time increased levels of free C3d and erythrocyte-bound C3d were observed. After 12 h free C3d was usually back to baseline level, but C3d on erythrocytes was still raised. These data indicate the formation of Streptokinase immune complexes in patients with high Streptokinase antibody levels, and show that these complexes are cleared rapidly from the circulation, leaving more persistent signs of complement activation. We conclude that free C3d is a good indicator of ongoing complement activation, whereas C3d on erythrocytes indicates that complement activation has recently taken place.

Correlation between serum immunoglobulin A concentrations and allergic manifestations in infants.

We studied the relationship of serum levels of IgA and IgE to allergic manifestations and otitis media in a cohort of 179 Icelandic children, aged 18 to 23 months. Only one of the infants had IgA deficiency (less than 50 micrograms/ml); all the others had IgA levels that were normal for their age. The children were divided into three groups according to their IgA levels (lowest 25%, intermediate 50%, highest 25%) and the clinical findings analyzed accordingly. The cumulative incidence of definite allergic manifestations was 37%. Asthma and otitis media were significantly more common among the infants with low normal IgA levels than among those with intermediate to high IgA levels. There was also a significant association between the severity of allergic manifestations and low IgA levels (p = 0.002). Children with detectable IgE (greater than or equal to 0.23 kilounit/L) had a higher incidence of atopic manifestations than did children in whom IgE was not detectable, but only a weak correlation was found between the occurrence and extent of allergic symptoms and increasing amounts of IgE beyond the 0.23 kilounit/L level. These findings suggest that atopic manifestations in infants may be more dependent on delayed maturation of IgA production than on overproduction of IgE.

Natural killer cell function and malignant cell phenotype in hairy cell leukaemia.

We have followed for 33 months the changes that occurred in natural killer (NK) cell numbers and activity in a patient (A) with hairy cell leukaemia (HCL), using a single cell assay and a microcytotoxicity assay. The composition of the peripheral blood mononuclear cell population and malignant cell phenotype were also analysed. During this period he received treatment with interferon and his grossly enlarged spleen was removed. Four further patients were also studied, two were splenectomized and all had received treatment with interferon. In four of the five patients studied there was an apparent link between low NK activity and presence of a tumour-infiltrated spleen, and in the fifth patient, who was aleukemic and had no splenomegaly, NK function was related to disease activity. There was no correlation between NK activity and the number of target binding (TB) cells in these five patients. IFN had little direct effect on overall NK activity, but the proportion of killing cells among TB cells was increased. Three patients showed binding of several cells to a single target. Further analysis revealed that in the patients most of the TB cells were not CD56-positive NK cells, in contrast to TB cells from normal subjects. In patient A a large proportion (84%) of TB cells were identified as malignant cells and in patient E 15% of TB cells were malignant cells. The phenotype of the malignant cells was: CD19+, HLA-DR+ and CD25(Tac)+, except for patient A. In this patient the hairy cells were positive for the NK marker CD56 as well as the monocyte marker CD14. Furthermore, a change occurred in phenotype as only later samples carried CD25. It is concluded that the level of NK function correlates closely with disease activity in HCL and that competitive target cell binding by malignant cells may be one cause of depressed NK-cell function in hairy cell leukaemia.

Disease manifestations in patients with isolated elevation of IgA rheumatoid factor.

In this retrospective study a comparison was made between the disease manifestations in patients with isolated elevation of IgA rheumatoid factor (RF) and patients with elevation of IgM RF. Of the 28 patients with isolated elevation of IgA RF, 14 (50%) had rheumatoid arthritis (RA) and 9 (32%) miscellaneous other inflammatory rheumatic disorders. It was found that 61% of these 23 rheumatic patients had disease manifestations from mucous membranes or secretory organs compared to 18% in the IgM RF positive group (p = 0.020). Patients with RA and an isolated elevation of IgA RF had more often mucosal or secretory symptoms than RA patients with elevation of IgM RF. We suggest that IgA RF may be a marker for activation of the mucosal or secretory immune system. The relationship between IgA RF and non-articular symptoms is discussed.

A flow cytometric assay for measuring complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes.

A flow cytometric assay (FCA) was developed to measure complement receptor 1 (CR1) and the complement fragments C3d and C4d on erythrocytes. It was possible to measure these parameters accurately with intra- and interassay coefficients of variation of 2.0% and 6.5% respectively. The method was able to discriminate between low and high levels of erythrocyte CR1, C3d and C4d. Comparison with a previously described RIA method gave excellent correlation coefficients with r2 values of 0.94, 0.93 and 0.91 for CR1, C3d and C4d respectively. The flow cytometric assay was used to measure CR1, C3d and C4d on the erythrocytes of 98 healthy individuals and the 95% upper limits for C3d and C4d were established. There was a wide distribution of CR1 levels amongst these individuals but their C3d and C4d levels were low and often not above background. The possible application of this method in clinical medicine is discussed.

Primary cytomegalovirus infection and gastric ulcers in normal host.

A 42-year-old woman presented with epigastric pain and vomiting. Upper gastrointestinal endoscopy revealed three gastric ulcers. Histologic examination of biopsies from the ulcers showed cytomegalovirus inclusion bodies. The appearance of IgM antibodies to cytomegalovirus indicated a recent and primary infection. Stored serum from her last pregnancy 17 months previously contained no cytomegalovirus antibodies. A thorough evaluation of her immune system revealed no abnormality. We are aware of only two other cases where seroconversion was documented in normal hosts. Cytomegalovirus infections in the gastrointestinal tract of normal hosts are very unusual but a common cause of morbidity in immunocompromised hosts. We believe that cytomegalovirus may have a role in the pathogenesis of gastrointestinal lesions in nonimmunocompromised patients.

The first Icelandic family with X-linked agammaglobulinaemia: studies of genetic markers and immune function.

This paper describes studies of genetic markers and immune functions in the first Icelandic family identified with X-linked agammaglobulinaemia (X-LA), including three affected brothers. The eldest brother was diagnosed at the age of 9 in 1963. He suffered repeated infections and died at the age of 23. The other two affected brothers, diagnosed at 6 years and 1 year of age, are alive and well on immunoglobulin replacement therapy at the ages of 32 and 24. All were typed for HLA, complement, and various other markers. Pedigree analysis suggests an X-linked segregation of the disease. Their serum IgG is maintained at normal levels on therapy. Several parameters of immune function were studied. The following results were obtained for the X-LA brothers: B cells are absent in their peripheral blood samples. T-cell numbers are normal, but monocytes are increased in numbers and activity. No immunoglobulin production could be elicited in vitro with PWM and no cells containing cytoplasmic Ig were detectable among PWM-stimulated blasts. Nevertheless the proliferative response was particularly vigorous, but the responding cells were shown to be exclusively T cells. No blast transformation could be achieved with EB virus. NK-cell activity was normal/high normal. Other cell-mediated immune functions were normal. In conclusion our data indicate that the differentiation of B cells is blocked in the two surviving X-LA brothers. They have survived for a longer time and in better health than is generally reported. Early diagnosis and adequate replacement treatment with Ig is clearly crucial. Vigorous non-specific immune mechanisms may help to compensate for the defective specific immunity.

IgG subclass response and opsonization of Streptococcus pneumoniae after vaccination of healthy adults.

Studies relating opsonization and IgG antibodies to Streptococcus pneumoniae have yielded contradictory results. This study compared changes in opsonization with IgG subclass response after vaccinating healthy subjects with a 23-valent pneumococcal vaccine. Total IgG and IgG subclass antibodies to pneumococcal polysaccharide types 8, 9, and 19 were measured by ELISA. Opsonic activity was assayed using 3H-labeled bacteria and polymorphonuclear leukocytes in different serum concentrations (5%-40%). A substantial postvaccination increase in total and subclass IgG antibody was observed in most subjects, although variations were seen. Postvaccination sera generally gave rise to enhanced opsonization, and a correlation was found between increases in antibody levels and opsonization. This correlation was closest for IgG1 and IgG4 and generally strongest at the lowest serum concentration, but weak or absent at the highest concentration. Thus, vaccination against S. pneumoniae stimulates a variable increase in specific opsonic activity in health persons that is best demonstrated when serum is a limiting factor in the opsonin assay.

Defective in vitro antibody production in response to pokeweed mitogen and influenza antigen in patients with Hodgkin's disease.

Cultures of peripheral blood mononuclear cells (PBM) from 33 patients with Hodgkin's disease, were stimulated in vitro with pokeweed mitogen (PWM) or influenza antigen. Impaired production of immunoglobulin (Ig) of one or more of the three main classes (IgG, IgM and IgA) in PWM stimulated cultures was found in 22 patients and in 11 patients no Ig of any class was produced. Antibody to influenza virus was detected in PWM stimulated PBM cultures in 13 of 14 normal individuals, but in only four of 25 patients with treated Hodgkin's disease though IgG was produced in 16 of 25. Influenza antigen induced anti-influenza antibody production in 10 of 12 cultures from normal individuals but in only two of 22 from patients. The results confirm our earlier report of defective antibody production in vitro by PBM from patients with Hodgkin's disease and indicate that polyclonally activated production of immunoglobulins of several classes is defective, though in vivo humoral immunity is normal.

Antibody-dependent cell-mediated cytotoxicity to Varicella zoster.

Antibody-dependent cell-mediated cytotoxicity (ADCC) against Varicella zoster (VZ) infected fibroblasts is described. ADCC requires antibody to VZ and is greater with heavily infected targets. It is not dependent on the immune status of the effector cells. The effector cells responsible for ADCC are present in sheep red cell (E) positive and E-fractions of peripheral blood, and in the G10 non-adherent population. The ADCC activity is present in microexudate non-adherent cells and in the adherent population to a lesser extent. The technique provides a means to study host defence against VZ infection in immune compromised individuals.

The effects of erythrocyte contamination on pokeweed mitogen induced immunoglobulin synthesis in man.

Red blood cell (RBC) contamination of cultures of peripheral blood mononuclear cells does not suppress pokeweed mitogen (PWM) induced immunoglobulin synthesis. At supra-optimal doses of PWM immunoglobulin production is enhanced in the presence of RBCs. The degree of RBC contamination is a factor to be considered in interpreting the results obtained with PWM induced Ig synthesis in disease.