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Background: Obesity is a heritable complex phenotype that can increase the risk of age-related outcomes. Biological age can be estimated from DNA methylation (DNAm) using various "epigenetic clocks." Previous work suggests individuals with elevated weight also display accelerated aging, but results vary by epigenetic clock and population. Here, we utilize the new epigenetic clock GrimAge, which closely correlates with mortality., Objectives: We aimed to assess the cross-sectional association of body mass index (BMI) with age acceleration in twins to limit confounding by genetics and shared environment., Methods and Results: Participants were from the Finnish Twin Cohort (FTC; n = 1424), including monozygotic (MZ) and dizygotic (DZ) twin pairs, and DNAm was measured using the Illumina 450K array. Multivariate linear mixed effects models including MZ and DZ twins showed an accelerated epigenetic age of 1.02 months (p-value = 6.1 × 10 -12 ) per one-unit BMI increase. Additionally, heavier twins in a BMI-discordant MZ twin pair (ΔBMI >3 kg/m 2 ) had an epigenetic age 5.2 months older than their lighter cotwin (p-value = 0.0074). We also found a positive association between log (homeostatic model assessment of insulin resistance) and age acceleration, confirmed by a meta-analysis of the FTC and two other Finnish cohorts (overall effect = 0.45 years, p-value = 4.1 × 10 -25 ) from adjusted models., Conclusion: We identified significant associations of BMI and insulin resistance with age acceleration based on GrimAge, which were not due to genetic effects on BMI and aging. Overall, these results support a role of BMI in aging, potentially in part due to the effects of insulin resistance., (© 2022 The Authors. Journal of Internal Medicine published by John Wiley & Sons Ltd on behalf of Association for Publication of The Journal of Internal Medicine.)

Background: A few studies have shown that both quick Sequential Organ Failure Assessment (qSOFA) score and cell-free DNA (cfDNA) have potential use as a prognostic marker in patients with infection. We studied these two markers alone and in combination to identify those emergency department (ED) patients with the highest risk of death., Methods: Plasma cfDNA level was studied on days 0 to 4 after admittance to the ED from 481 culture-positive bloodstream infection cases. The qSOFA score was evaluated retrospectively according to Sepsis-3 definitions. The primary outcome was death by day 7., Results: CfDNA on day 0 was significantly higher in nonsurvivors than in survivors (2.02 μg mL -1 vs. 1.35 μg mL -1 , P < 0.001). CfDNA level was high (>1.69 μg mL -1 ) in 134 (28%) of 481 cases, and the qSOFA score was ≥2 in 128 (28%) of 458 cases. High cfDNA and qSOFA score ≥2 had 70% and 77% sensitivity and 76% and 76% specificity in predicting death by day 7, respectively. High cfDNA alone had odds ratio (OR) of 7.7 (95% CI 3.9-15.3) and qSOFA score ≥2 OR of 11.6 (5.5-24.3), but their combination had OR of 20.3 (10.0-41.4) in predicting death by day 7 when compared with those with low cfDNA and qSOFA score <2. Amongst the five cases with the highest cfDNA levels, there were three patients with severe disseminated intravascular coagulation., Conclusion: CfDNA and qSOFA score can be used independently to identify those bacteraemia patients at high risk of death, and combining these two markers gives additional advantage., (© 2018 The Association for the Publication of the Journal of Internal Medicine.)

Objectives: Urokinase-type plasminogen activator receptor (uPAR) is expressed on a variety of different immune cells and vascular endothelial cells during inflammation. Previous studies indicate that a high plasma concentration of the soluble form of the receptor (suPAR) predicts poor outcome in infectious diseases., Design: A prospective cohort study., Subjects and Methods: Plasma suPAR levels were measured in 132 patients with bacteraemia caused by Staphylococcus aureus, Streptococcus pneumoniae, ß-haemolytic streptococcae or Escherichia coli using a commercial enzyme-linked immunosorbent assay (ELISA). Values were measured on days 1-4 after a positive blood culture, on days 13-18 and on recovery., Results: The maximum suPAR values on days 1-4 were markedly higher in nonsurvivors compared to survivors (15.8 vs. 7.3 ng mL(-1) , P < 0.001) and the area under the receiver operating characteristic curve (AUC(ROC) ) in the prediction of case fatality was 0.84 (95% confidence interval (CI) 0.76-0.93, P < 0.001). At a cut-off level of 11.0 ng mL(-1) , the sensitivity and specificity of suPAR for fatal disease was 83% and 76%, respectively. A high level of suPAR (≥ 11 ng mL(-1) ) was associated with hypotension (mean arterial pressure < 70 mmHg) (odds ratio (OR) 6.5; 95% CI 2.9-14.6) and high sequential organ failure assessment score (≥ 4) (OR 9.3; 95% CI 4.0-21.9). A high suPAR level remained an independent risk factor for case fatality in a logistic regression model adjusted for potential confounders., Conclusion: Plasma suPAR level is a sensitive and specific independent prognostic biomarker in patients with bacteraemia., (© 2011 The Association for the Publication of the Journal of Internal Medicine.)

Background: Serum amyloid A (SAA) is a sensitive marker of inflammation and its elevation has been implicated in obesity and in cardiovascular disease, yet data on its regulation in young adults or on its role in early atherosclerosis is scarce. We investigated which factors explain the variation in SAA and analysed whether SAA could be associated with preclinical atherosclerosis., Methods: Serum amyloid A levels were measured in participants of the Cardiovascular Risk in Young Finns Study (n = 2280, n = 1254 women, n = 1026 men). Correlates and determinants of SAA were analysed and the effect of SAA on subclinical atherosclerosis, measured as intima-media thickness (IMT) and carotid artery compliance, was evaluated with risk-factor adjusted models., Results: Serum amyloid A correlated directly and independently of BMI with C-reactive protein (CRP), waist circumference and leptin in both sexes, with total cholesterol, LDL cholesterol and ApolipoproteinA1 (ApoA1) in women and with triglycerides, insulin levels and insulin resistance in men. Use of combined oral contraceptives and intrauterine device was also associated with SAA levels. Determinants for SAA included CRP, leptin and ApoA1 in women, and CRP, leptin and HDL cholesterol in men. SAA levels correlated with carotid compliance in both sexes and with IMT in men, yet SAA had no independent effect on IMT or carotid compliance in multivariable analysis., Conclusions: Serum amyloid A was associated with several metabolic risk factors but was not an independent predictor of IMT or carotid artery compliance. Further longitudinal studies will show whether SAA holds a prognostic value as a risk marker, analogously to CRP.

Background: Total serum IgE is regulated by both environmental and genetic factors. Association and linkage studies have suggested a role of CD14-159C>T polymorphism in the regulation of serum total IgE, but the results have been contradictory. It seems that gene-environment interactions are involved in this regulation., Objective: The aim of this study was to examine the possible gene-environment interactions among Toxoplasma gondii, Helicobacter pylori, CD14-159C>T and Toll-like receptor (TLR) 4+896A>G polymorphism on serum total IgE. For this study, we expanded the scope of our earlier comparison of allergic sensitization and microbial load between Finland and Russian Karelia by studying the CD14-159C>T and TLR4+896A>G polymorphism in a cohort of Russian Karelian children., Methods: For this study, CD14-159C>T and TLR4+896A>G polymorphisms were analysed in 264 healthy Russian Karelian children. Serum total IgE levels and H. pylori and T. gondii antibodies were also measured., Results: We constructed a multiway anova model to analyse the gene-environment interactions among T. gondii seropositivity, H. pylori seropositivity, CD14-159C>T and TLR4+896A>G polymorphisms on serum total IgE. The model showed that there was an interaction between the CD14-159 allele T carrier status and H. pylori antibodies on serum total IgE (P=0.004). No other interactions were found., Conclusion: Our results further emphasize the role of gene-environment interaction in the regulation of serum total IgE.

Mannose-binding lectin (MBL) insufficiency caused by point mutations in the MBL2 gene has been associated with increased susceptibility to bacteraemic infections. We here investigated the effect of MBL2 polymorphisms on the susceptibility and clinical course of bacteraemia. The study cohort comprised 145 patients with bacteraemia and 400 controls. In the case of patients with bacteraemia, laboratory findings and clinical data were registered on admission and during six consecutive days. MBL2 structural polymorphisms at codons 52 (CGT-->TGT; designated D or O), 54 (GGC-->GAC; B or O) and 57 (GGA-->GAA; C or O) in exon 1 of the MBL2 gene and promoter region polymorphisms at position -221 (G-->C, designated Y or X alleles) were determined. No difference in MBL2 genotype frequencies between the bacteraemic patients and controls was detected, and MBL2 genotype had no independent effect on mortality, nor disease severity. However, smoking proved a significant risk factor for Gram-positive (Staphylococcus aureus, Streptococcus pneumoniae or beta-haemolytic streptococci) bacteraemia in patients carrying the variant O allele (53% current smokers in Gram-positive bacteraemia patients compared with only 21% in controls, odds ratios 4.2, 95% confidence intervals 2.0-9.0; P < 0.001), while it did not have an effect in those homozygous for the A allele. The same effect was not detected in Escherichia coli bacteraemia. In conclusion, MBL2 genotypes representing MBL insufficiency were not associated with the overall risk of bacteraemia or disease severity, but smoking in carriers of the structural variant O allele may have a deleterious effect increasing the risk of Gram-positive bacteraemia.

Objective: The aim of this study was to investigate whether polymorphism of the mannose-binding lectin 2 (MBL2) gene is related to the occurrence of systemic AA amyloidosis in patients with rheumatoid arthritis (RA)., Methods: MBL2 structural gene polymorphisms at codon 52 (CGT-->TGT, Arg-->Cys; D), codon 54 (GGC-->GAC, Gly-->Asp; B) and codon 57 (GGA-->GAA, Gly--> Glu; C), and MBL2 promoter region polymorphism at position -221 (G-->C) were examined in 57 patients with RA complicated by biopsy-proven reactive amyloidosis and 51 control RA patients without amyloid., Results: A strong association was found between the presence of a structural MBL2 gene variant O (B, D or C) and the occurrence of amyloidosis in RA patients: 29 of 57 (50.9%) of the RA patients with amyloid had a variant allele compared with 12 of 51 (23.5%) of the RA patients without amyloid (OR 3.37, 95% CI 1.47-7.72; P = 0.004)., Conclusion: We conclude that variant MBL2 structural genotype constitutes a significant risk factor for reactive amyloidosis in RA and that the increased risk is probably related to MBL-mediated impairment of mononuclear phagocyte function.

Abstract Mechanisms induced by tryptophan (trp) catabolism are important in the regulation of both normal and pathogenetic immune responses. The key enzyme is indoleamine-pyrrole 2,3-dioxygenase (EC 1.13.11.42) (IDO) which converts trp to kynurenine (kyn), the main toxic metabolite. It is known that interferon-gamma (IFN-gamma) is able to activate IDO. We wanted to analyse whether the strength of this mechanism would be under genetic control. To this end, we analysed the IFN-gamma+874(T/A) genotypes, which are known to have an effect on IFN-gamma production, of 309 healthy blood donors and correlated these to the levels of trp and kyn in their blood. The data obtained demonstrate that the presence of the high producer T allele was associated with increased IDO activity (i.e. elevated kyn and kyn/trp levels), but this effect was observed only in females. These data show that trp catabolism is genetically controlled by the IFN-gamma gene and may thus be operative in those disease conditions associated with the polymorphisms of the IFN-gamma gene.

Objective: In amyloid A (AA) amyloidosis the receptor for advanced glycation end products is a target for the circulating amyloid precursor protein (SAA) resulting in upregulation of the proinflammatory cytokine pathway. Besides inducing hepatic SAA synthesis the interleukin-1 cytokine family is involved in the regulation of haematopoiesis. We therefore studied the relationship between the circulating levels of interleukin-1beta (IL-1beta) and interleukin-18 (IL-18), a new member of the IL-1 complex, as well as polymorphisms within the IL-1 cluster with the occurrence of anaemia in patients with AA amyloidosis., Design, Setting and Subjects: The study included 54 adult patients with biopsy-proven reactive amyloidosis allocated into three groups on the basis of haemoglobin (Hb) level: group I included all patients with Hb < 110 g L(-1) (n = 16); group II patients (Hb > 110 g L(-1), n = 16) were selected to match group I patients with respect to sex, age, underlying disease (seropositive, erosive rheumatoid arthritis) and renal function; and group III patients (n = 38) represented all patients (unselected) with Hb > or = 110 g L(-1). Gene polymorphisms were studied by polymerase chain reaction restriction length assay and included the base exchange at position-889 of the IL-1alpha gene, the polymorphic region at position-511 and the polymorphic locus at exon 5, position +3954 of the IL-1beta gene, as well as the IL-1 receptor antagonist (IL-1Ra) exon 2 polymorphism caused by the 86-bp tandem repeats. Plasma IL-1beta, IL-1alpha, IL-18, IL-1 Ra, SAA, ferritin, soluble transferrin receptor and erythropoietin levels were studied by enzyme immunoassays., Results: Circulating IL-beta and IL-18 were significantly raised in the anaemic patients with AA amyloidosis when compared with group II patients (matched, Hb > 110 g L(-1)) as well as group III patients (nonmatched, Hb > or = 110 g L(-1)). A significant inverse relationship was found between IL-1beta and haemoglobin levels, as well as between IL-18 and haemoglobin levels. The frequency of allele 2 (T) of the IL-1beta-511 promoter gene was significantly increased and that of allele 1 (C) decreased in anaemic amyloid patients (group I) when compared with group II and III patients. Circulating IL-1beta levels tended to be higher amongst the IL-1beta-511 allele 2 carriers than amongst the noncarriers, as well as amongst the anaemic amyloid patients filling all criteria of anaemia of chronic disease., Conclusion: The occurrence of anaemia in patients with AA amyloidosis is associated with allele 2 (T) of the IL-1beta-511 promoter gene and elevated levels of circulating IL-1beta and IL-18. In AA amyloidosis the raised cytokine levels may generate a vicious cycle leading to accelerated amyloidogenesis, suppression of erythropoiesis and aggravation of the underlying inflammatory disorder.

The immunoglobulin (Ig) plasma levels are known to be, at least partially, genetically regulated, but all the genes involved are not known. Interleukin-1 (IL-1) is a potent proinflammatory cytokine able to serve as an adjuvant for immune responses. IL-1alpha gene is polymorphic, and at least one of the polymorphisms has been identified in the 5' regulatory region of the promoter, a biallelic base exchange (C-->T) at position -889. We set out to study whether the IL-1alpha genotype might contribute to the genetic component seen in the steady-state antibody levels of healthy individuals. Four hundred healthy blood donors (218 males and 182 females) were genotyped, and the plasma levels of IgM, IgG as well as IgG subclasses were measured. An association was found between IgG3 plasma levels and the IL-1alpha genotype; the 1.1 homozygotes had increased IgG3 levels compared with the 1.2 heterozygotes (P < 0.001 in males and P = 0.04 in females, Mann-Whitney U-test). A similar significant association was also found between IgM plasma levels and the IL-1alpha genotype in males, but it was no longer present in females; the 1.1 homozygotes had higher IgM levels than the 2.2 homozygotes (P = 0.03, Mann-Whitney U-test). The data suggest that IL-1alpha-mediated signals are critical for IgG3 and IgM responses, which are induced by thymus-independent antigens and are important in activating complement.

Background: IL-10 has several functional effects relevant to asthma. It can modulate IgE production and induce apoptosis in eosinophils. Polymorphisms of IL-10 gene have been shown to affect IL-10 production., Objective: To establish whether IL-10 polymorphisms are associated with asthma and phenotype-related characteristics., Methods: The frequency of three single base exchange polymorphisms (at positions - 1082, - 819 and - 592) and corresponding haplotypes of the IL-10 gene were analysed in 245 adult asthmatic subjects and 405 controls using PCR and restriction fragment length polymorphism (RFLP). The data were assessed for correlations with the eosinophil count, serum IgE and lung function., Results: The IL-10 haplotype frequencies were similar in asthmatics and controls. Eosinophil count median was 2.0- to 3.2-fold higher among asthmatics with rare ATA/ATA genotype than in asthmatics with other genotypes. No such difference was seen in the control group. When analysed by IL-10 haplotype carrier state and gender, male asthmatics with ATA haplotype had 2.8-fold higher serum IgE than those without ATA. A converse association was found in male controls with ATA haplotype, who had 1.9-fold lower serum IgE than their ATA-negative counterparts. The high IL-10-producing GCC haplotype was associated with impaired lung function in smoking male controls while in asthmatics no clear effect on lung function was found with any of the haplotypes studied., Conclusion: These results suggest that the eosinophil counts and serum IgE are differently regulated by IL-10 genotype in asthmatic and in normal subjects. However, IL-10 polymorphism is not related to susceptibility in asthma.

Oral Lactobacillus rhamnosus GG ingestion for 5 days to 4 weeks has been shown to alleviate clinical symptoms of gastrointestinal inflammation and atopic dermatitis. To determine whether oral Lactobacillus rhamnosus GG may act by generating immunosuppressive mediator in atopic children. Lactobacillus rhamnosus GG (ATCC 53103) at a daily dose of 2 x 1010 cfu was added for 4 weeks to the diets of nine children (mean age, 21 months) with atopic dermatitis. Blood and faecal samples were collected before supplementation and at early (2 weeks) and late stage (4 and 8 weeks from the beginning). The concentrations of interleukin-6 (IL-6), IL-10, IL-12, tumour necrosis factor-alpha (TNFalpha) and interferon-gamma (IFNgamma) in sera, as well as the production of IL-2, IL-4, IL-10 and IFNgamma in mitogen-induced peripheral blood mononuclear cells, were assessed. Secretory IgA and TNFalpha were also determined in faeces. The serum IL-10 concentration differed significantly between before, early and late samples (P < 0.001) due to the elevation of serum IL-10 in the later phase of oral Lactobacillus rhamnosus GG ingestion. The enhancement of IL-10 production in mitogen-induced cultures preceded the rise in serum IL-10. The enhanced IL-10 generation in vivo substantiates the anti-inflammatory properties of specific probiotic bacteria strains, and provides an additional reason for considering such treatments for patients with intestinal inflammation.

Activated T cells, with their secretion of cytokines, probably play an important role in the pathogenesis of mucosal lesions in coeliac disease (COD) and the prominence of a T-helper (Th)1-type cytokine pattern has been reported. As the process of immunological activation in the jejunal mucosa in active CoD has been shown to also cause some differences in peripheral blood lymphocyte populations, we sought to establish any changes in the Th 1/Th2 balance in peripheral blood of patients, at different stages of CoD, relative to healthy individuals. Twenty-two CoD patients and 10 healthy controls were included in the study. The Th1/Th2 balance was examined both in resting cells and after polyclonal stimulation using two different methods: intracytoplasmic cytokine contents were measured using an intracellular staining method and three-colour flow cytometry and cytokine contents of cell culture supernatants were measured using traditional enzyme-linked immunosorbent assays (ELISAs). Interferon-gamma (IFN-gamma)-producing cells (Thl) were as prominent in untreated CoD patients and treated CoD patients as in healthy controls, while cells fitting a Th2 or ThO-type cytokine pattern were few in all groups. In ELISA assays, Th1 type (IFN-gamma or interleukin (IL)-2) cytokines were again prominent in all study groups but no statistically significant differences were found in IFN-gamma, IL-4 or IL-2 levels among the three groups. These results suggest that the increased shift towards a Th1 response is mainly restricted to the actual site of inflammation and that circulating T cells do not show a similar response, presumably because activated cells in peripheral blood are too few. Further research on cytokine profiles measuring T-cell activation in CoD should be focused on the actual tissue of inflammation.

Epstein-Barr virus (EBV) seronegativity is rare in adults. To examine whether genetic differences would explain this, we studied the genetic polymorphisms of the genes of the interleukin-1 (IL-1) complex in seronegative adults. These cytokines (i.e. IL-1alpha, IL-1beta and IL-1 receptor antagonist, IL-1RA) regulate, in several ways, the inflammatory reactions of the body. In each of these genes there are polymorphic sites and the various alleles differ in their frequency in several diseases of inflammatory nature. In 400 healthy blood donors (from 18 to 60 years of age) there were 20 (5%) seronegative persons. The frequency of allele 2 of the IL-1beta gene (base exchange polymorphism at position -511 from the transcriptional start site) was decreased in the seronegative patients (0.20 versus 0.42 in the seropositive patients, P < 0.05, chi2-test). Moreover, the frequency of allele 2 of the IL-1RA (polymorphism defined by variable numbers of 86-bp repeats in intron 2) was slightly, but not significantly, decreased in the seronegative patients. Alleles of these two loci are known to be associated, but in the seronegative patients this association was abnormal: 11 out of 20 (55%) were of the IL-1RA-2 negative/IL-1beta-2 negative type, while of the seropositive patients, 25% were of this type (P < 0.01, chi2-test). These data suggest that immunological differences, depending on cytokine gene polymorphisms, regulate the resistance to EBV infection.

The capacity of neonatal T cells to secrete interleukin-2 (IL-2) has been reported to be variable. We analysed IL-2 production in purified neonatal and adult T cells using polyclonal activator phorbol ester + calcium ionophore (PDBu + iono) or receptor-mediated anti-CD3/anti-CD3+ anti-CD28 stimulation. PDBu + iono induced equally high IL-2 levels in both groups and, when stimulated with plate-bound anti-CD3 monoclonal antibody (mAb), the IL-2 secretion by neonatal cells was undetectable and adult cells produced low amounts of IL-2 (mean 331 +/- 86 pg/ml). The addition of anti-CD28 mAb to anti-CD3-stimulated cells markedly increased IL-2 production in both cell types, but levels of IL-2 in neonatal T cells remained clearly lower than those of adult T cells (respective mean values: 385 +/- 109 pg/ml and 4494 +/- 1199 pg/ml). As NF-kappa B is a critical transcription factor in the control of IL-2 expression, we next analysed its nuclear translocation in neonatal and adult T cells using the electrophoretic mobility shift assay and, because induction of reactive oxygen intermediates (ROI) is required for the activation of NF-kappa B, we also analysed levels of intracellular ROI in these cells using the ROI-reactive fluorochrome DCFH-DA and flow cytometry. In neonatal T cells NF-kappa B activation and ROI formation after anti-CD3 stimulation were low compared with adult T cells and, although addition of anti-CD28 mAb increased induction of NF-kappa B and ROI formation, levels similar to those of adults were not achieved. After PDBu + iono stimulation, the cells showed similar ROI formation and IL-2 secretion. Our results suggest that reduced IL-2 production by neonatal T cells is specific for anti-CD3 and anti-CD3+ anti-CD28-mediated stimulation and that these activators cannot effectively activate the ROI-NF-kappa B signalling pathway in neonatal T cells.

The genes of the interleukin-1 (IL-1) complex code for three proteins: IL-1alpha, IL-1beta and the IL-1 receptor antagonist (IL-1RA). Each of these genes is polymorphic and there is increasing evidence that certain alleles are associated with increased susceptibility to a given disease of inflammatory nature. In the IL-1beta gene there are two base-exchange polymorphisms in positions -511 and +3953, and IL-1RA gene has a penta-allelic polymorphic site in intron 2 containing variable numbers of an 86-bp tandem repeat sequence. As the IL-1beta/IL-1RA ratio may be critical in the regulation of inflammation, we examined whether there are allelic associations between these loci (thus suggesting co-ordinate regulation) and whether these have an effect on the in vitro production of IL-1beta. We found that the IL-1RA allele 2 (IL1RN*2) is associated with the presence of allele 2 of the IL-1beta gene (position -511) and with the absence of allele 2 of the IL-1beta gene (position +3953). Mononuclear cells from carriers of allele 2 (position -511) and non-carriers of allele 2 (position +3953) had a slight, but non-significant, elevated capacity to produce IL-1beta in vitro. However, IL-1RA allele 2 strongly increased in vitro production of IL-1beta, regardless of the presence or absence of these alleles. Taken together, these data suggest that the known allelisms in the IL-1beta gene are not major regulators of the in vitro IL-1beta production, but the IL-1RA allele 2 (or an unknown allele strongly associated with it) has a decisive role.

Interferon gamma (IFN-gamma) production as a measure of cellular sensitization was studied by detection of the cytokine in culture supernatant by enzyme immunoassay (EIA) and by measuring cellular mRNA using the reverse transcriptase polymerase chain reaction (RT-PCR) method. These assays were compared to the standard lymphocyte proliferation assay as a marker of T cell responsiveness to foreign antigens. When blood donors seropositive for herpes simplex virus (HSV) were compared to seronegative donors, all measurements of cellular sensitization separated the groups without overlap. There were significant correlations between the IFN-gamma mRNA titre and the secreted IFN-gamma (r = 0.57, P = 0.03), and the proliferative response and the secreted IFN-gamma (r = 0.78, P = 0.001), as well as between the IFN-gamma mRNA titre and the proliferative response (r = 0.78, P < 0.001). When tetanus toxoid (TT) responses were studied in immunized subjects, a wide range of responsiveness could be seen and correlation between various measurements was poor. However, constant individual levels of the cytokine production were demonstrated. Six people who had received their last TT booster vaccination more than 5 years ago were revaccinated and repeatedly studied. An increase in the levels of produced IFN-gamma could be seen in all subjects and two who lacked a lymphocyte proliferation response developed it after revaccination.

Background: A major feature of atopic dermatitis (AD) is the propensity to generate IgE to environmental antigens. Despite extensive information on IgE dysregulation in AD, the nature of immune responses to ingested allergens is poorly characterized., Objective: To determine the clinical and immunological responses to food in AD patients., Methods: To characterize the type and timing of clinical reactions to oral cow milk, 83 AD patients aged 2 to 60 months were subjected to double-blind placebo-controlled food challenge (DBPCFC). IFN-gamma and IL-4 production by their peripheral blood mononuclear cells (PBMC) was determined before and after DBPCFC., Results: Of 50 patients positive to DBPCFC. 46% manifested exanthematous-type immediate-onset reactions and 54% eczematous-type late-onset reactions. In either group, the production of IL-4 and IFN-gamma by Concanavalin A-stimulated PBMC was comparable before and after DBPCFC. For immediate-reacting patients, the median IFN-gamma production by milk-stimulated PBMC was 11.5 (4.2-17.2) pg/ml as against 2.3 (0.2-5.7) pg/ml by unstimulated PBMC, P = 0.0008 before DBPCFC, and 4.6 (2.8-10.3) pg/ml. vs 4.2 (1.7-9.0) pg/mL, p = 0.40, correspondingly after DBPCFC., Conclusion: Before DBPCFC, immediate-reacting but not late-reacting patients were found to be capable of allergen-specific IFN-gamma production in vitro, indicating the heterogeneity in AD patients. After DBPCFC, the IFN-gamma generation abolished, indicating the effect of oral allergen exposure on IFN-gamma producing responses of AD patients.

The molecular background of the defects in the immune reactivity of human neonates has not been fully elucidated. As the NF-kappa B transcription factor has a central role in the control of transcription of several genes involved in immune and inflammatory responses, the authors have analysed the activation of NF-kappa B in human umbilical cord T lymphocytes. The activity was tested by quantitating the nuclear proteins binding to an oligonucleotide containing the consensus kappa B binding sequence (electrophoretic mobility shift assay). The data obtained demonstrate that phorbol dibutyrate/calcium ionophore A23187 (PDBu/iono) combination induced a clearly higher nuclear translocation of NF-kappa B in neonatal than adult T cells. This higher NF-kappa B activity was restricted to the CD4+ T-cell subset. Analysis of the nuclear extracts with antibodies directed against the major components of NF-kappa B the p50 and RelA (p65) proteins, indicated that the composition of NF-kappa B was similar in neonatal and adult cells. These results suggest that neonatal T cells are exposed to oxidative stress-inducing signals during delivery and/or are inherently more sensitive to NF-kappa B activating signals than adult T cells.

Cyclic AMP (cAMP)-dependent protein kinase A (PKA) is known to have both negative and positive effects on the activation mechanisms of T lymphocytes. The authors have analysed the effect of increased cAMP on the activation of NF-kappa B transcription factor. This factor controls the expression of several genes (e.g. IL-2 and IL-2 receptor) involved in the activation and proliferation of T cells. The authors found that elevation of intracellular cAMP in Jurkat T leukaemia cells activated with phorbol ester (PDBu)/calcium ionophore (A23187) increased the DNA-binding of NF-kappa B as detected by the electrophoretic mobility shift assay (EMSA). Analysis of the subunit composition of the DNA-binding complex indicated that the amount of c-Rel was enhanced while RelA was decreased. Analysis of the effect of elevated cAMP on the degradation of I kappa B-alpha and I kappa B-beta did not reveal an essential change in degradation kinetics of these inhibitor proteins. The elevation of cAMP did not increase the synthesis of c-Rel, but it enhanced the nuclear localization of this protein. Transfection of Jurkat cells with a plasmid kB/TK10-CAT indicated that the increased DNA-binding of c-Rel containing complexes seen in EMSA was also functional. These data imply that the strong and long-lasting c-Rel nuclear localization and DNA-binding induced by protein kinase A is not due to increased c-Rel synthesis or enhanced degradation of the I kappa B inhibitors. Therefore, a direct phosphorylation of the c-Rel protein is the most plausible explanation for these observations. Taken together, these results suggest that cAMP is able to regulate the expression of NF-kappa B-dependent genes in T cells by modifying the composition and subunit activity of NF-kappa B.

A prerequisite for systemic hyporesponsiveness to dietary antigens is their processing in the gut. This study investigated whether bovine caseins degraded by enzymes of an intestinal bacterial strain, Lactobacillus GG (ATCC 53103), could regulate the cytokine production by anti-CD3 antibody-induced peripheral blood mononuclear cells of 14 atopic patients, aged 5-29 (mean, 16) months. Purified casein up-regulated the interleukin-4 and interferon-gamma production, P = 0.008 and P = 0.008, respectively. Conversely, Lactobacillus GG-degraded casein down-regulated the interleukin-4 production, P = 0.003, with no effect on interferon-gamma. These results indicate that intestinal bacteria may modify immunomodulatory properties of native food proteins and introduce a promising tool to provide protection from potentially harmful dietary antigens at a young age.

Protein kinase C (PKC)-activating phorbol esters are known to induce the expression of several genes in monocytic cells. As the effect of serine-threonine kinases, such as PKC, is often counteracted by specific protein phosphatases, we have now examined the role of phosphatases in the regulation of the phorbol ester (PMA)-induced interleukin-1 beta (IL-1 beta) gene expression in the THP-1 monocytic leukaemia cell line. Okadaic acid (OA) is a potent tumour promoter, the function of which is based on its activity to inhibit the serine/threonine specific phosphatases 1 and 2A (PP1 and PP2A, respectively). Thus, it mimicks or potentiates the action of PKC activators in several cell types. Our data demonstrate that alone OA induced a very weak expression of IL-1 beta mRNA, but it strongly enhanced the PMA-induced IL-1 beta expression. To analyse the site of action of OA, the cells were transiently transfected with a chloramphenicol acetyl transferase (CAT)-reporter plasmid containing the AP-1 binding site as the enhancer. Alone, OA was a weak inducer of CAT-activity in these cells, but again it strongly enhanced the PMA-induced response. Similar data were obtained with cells transfected with a reporter plasmid containing the PMA-responsive element (containing a putative AP-1 binding site) of the IL-1 beta gene. Thus, these data indicate that the PMA-induced AP-1 enhancer activity, which is required for the expression of the IL-1 beta gene, is controlled in these cells by PP1 and/or PP2A. As OA did not synergize with PMA in the induction of expression of genes encoding the AP-1 proteins (c-fos, c-jun, junB), it is likely that OA potentiates the AP-1 enhancer activity by its effect on protein phosphorylation.

The role of elevated intracellular calcium concentration [Ca2+]i in the LPS-induced activation of interleukin-1 beta (IL-1 beta) production was examined in cells representing different stages of myeloid differentiation (undifferentiated monocytic leukaemia cell line THP-1, THP-1 cells induced to adherent, macrophage-like cells by phorbol ester treatment and normal peripheral blood-derived adherent monocytes). LPS did not elevate the [Ca2+]i as measured by the Fura-2 fluorescence technique. When these cells were stimulated with LPS in the presence of the calcium ionophore A23187, a clear increase in the IL-1 beta protein production was observed in the undifferentiated THP-1 cells but not in the more differentiated cell types. This ionophore-induced increase was also seen in the IL-1 beta mRNA levels. Thus these data confirm the previous findings demonstrating that elevation of [Ca2+]i is not involved in the LPS-dependent signal transmission. However, the LPS-induced signals are greatly potentiated by the elevated [Ca2+]i, but only in undifferentiated monocytic cells.

The capability of elevated intracellular cyclic AMP concentration to activate IL-1 gene expression and protein production was examined in human peripheral blood monocytes. In accordance with previous studies it was observed that the transiently elevated cyclic AMP (induced either with prostaglandin E2 or with the direct adenylate cyclase activator, forskolin) was not a sufficient signal to activate IL-1 production. However, if the degradation of cyclic AMP was inhibited with isobutyl-methyl-xanthine (IBMX), IL-1 production was strongly activated. This prostaglandin E2 plus IBMX effect could also be mimicked with high concentrations of the cell permeant structural cyclic AMP analogue, dibutyryl cyclic AMP. The cyclic AMP-induced IL-1 production differed in some aspects from the bacterial lipopolysaccharide-induced IL-1 production: (1) the kinetics of both IL-1 gene expression and protein production was much slower; (2) the IL-1 beta gene expression was superinducible by inhibiting the protein synthesis with cycloheximide. Thus these data suggest that prolonged elevation of cyclic AMP is alone a sufficient signal to activate IL-1 production.

Interleukin-1 beta (IL-1 beta) is a cytokine produced mainly by activated monocytes though the mechanism by which it is released is still unknown. Elevation of intracellular cyclic adenosine monophosphate (cAMP) is considered an important down-regulative signal in the production of IL-1 beta in lipopolysaccharide (LPS)-induced monocytes. In this study we show that in LPS-activated human monocytes, elevated cAMP concentrations (induced by either prostaglandin E2, forskolin or dibutyrylcyclic AMP) affected specifically secretion of IL-1 beta; the amount of secreted IL-1 beta was clearly reduced whereas the cell-associated level remained unchanged. TNF-alpha, a normal secretory protein, was used as a control. Cyclic AMP also inhibited TNF production by monocytes, but the decrease was of the same magnitude in the extracellular and intracellular compartments. Thus, the down-regulative effect of cAMP on the production of these monokines is clearly different.

The production and mRNA expression of IL-1 alpha and IL-1 beta by human monocytes was examined after two different stimuli, a protein kinase C (PKC) activator phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS). LPS induced production of high levels of both IL-1 alpha and IL-1 beta protein (quantitated with type-specific ELISA assays), while after PMA stimulation only IL-1 beta protein could be detected. The IL-1 alpha and IL-1 beta mRNA levels quantitated by Northern blotting were in line with the respective protein levels and nuclear run off analysis revealed that PMA did not activate the IL-1 alpha transcription. The production of the IL-1 alpha and IL-1 beta protein as well as the mRNA expression could be inhibited with protein kinase inhibitor H7, but not with HA1004, indicating that PKC activation is essential for the activation of these genes. Thus these data indicate that PKC activation alone is sufficient for the induction of the IL-1 beta gene, but some additional signals (provided by LPS) are required for the activation of the IL-1 alpha gene.

Little is known about the non-antigen-specific signals delivered to T cells by dendritic cells (DC). Because several monocyte-derived factors like interleukins 1 alpha, 1 beta, and 6 (IL-1 alpha, IL-1 beta, and IL-6) enhance the T-cell proliferative responses, we studied the production of the above-mentioned cytokines by DC separated from human peripheral blood. The intracellular expression of the proteins (IL-1 alpha and IL-6) was studied at a single-cell level using an immunolabelling technique. The supernatants and cell lysates were studied with ELISA (IL-1 alpha and IL-1 beta). Northern blotting analysis was used to quantitate the mRNA levels. Several approaches were taken to stimulate the production of IL-1 alpha, IL-1 beta, and IL-6 by DC. These included the incubation of the DC in the presence of either LPS, rIL-1, or monoclonal anti-HLA-DR antibody, or the stimulation of cells with resting allogeneic T cells. None of the stimuli was able to induce the production of IL-1 alpha, IL-1 beta, or IL-6 by DC, whereas LPS-stimulated monocytes were strong producers of these mentioned cytokines and expressed the respective mRNA. Thus we concluded that IL-1 alpha, IL-1 beta, and IL-6 are primarily monocyte-derived factors and that these factors are not needed or produced during the activation of resting T cells by DC.

Activation of resting T cells to proliferate is usually accompanied by their expression of interleukin 2 receptors (IL-2R) and secretion of (IL-2). We studied the mechanisms by which human blood-derived dendritic cells (DC) and monocytes induce IL-2R and stimulate IL-2 secretion in autologous and allogeneic mixed luecocyte reaction (auto- and allo-MLR, respectively). We found that only DC were fully effective as stimulator cells in MLR. DC stimulated both autologous and allogeneic T cells to express high-affinity IL-2R, secrete IL-2, and vigorously proliferate in MLR. The stimulatory properties of monocytes were more complicated: although they stimulated the proliferation in allogeneic MLR, the proliferation rates, duration, and amount of IL-2 secretion were different than in DC-induced MLR. Autologous T cells did not proliferate in response to monocytes, but were induced to express the low-affinity IL-2R. If the cultures were supplemented with exogenous recombinant IL-2, the proliferative responses to DC and monocytes in auto- and allo-MLR were of the same magnitude, indicating that the responsiveness to IL-2 was stimulated by both the stimulator cells. The stimulator cell number was important, since large numbers of monocytes, but not of DC, were suppressive to the proliferative responses. Thus, we concluded that the higher capacity of DC, as compared to monocytes, to stimulate T-cell proliferation is based primarily on the more efficient stimulation of IL-2 secretion.

The regulatory role of adult thymocytes in the in vitro cytotoxic cell formation against TNP-coupled syngeneic spleen cells was studied. Thymocytes are known to amplify the cytoxic response of lymph node cells against allogeneic cells. This kind of synergism was not found in the response against TNP-coupled cells; on the contrary, thymocytes had a clear suppressive effect. Thymocytes inducing this suppression must be present already at the beginning of the in vitro response. Mitotically blocked (mitomycin-C treated) thymocytes but not heat killed or lysed cells were also capable to suppress anti-TNP cytotoxicity.

Most of the natural killer (NK) cells, recently derived from the bone marrow (14 days after injection of bone marrow cells into lethally irradiated mice), express the Thy-1 antigen; after some period of time (by day 28) a normal proportion (50%) of Thy-1+ NK cells is found. This study demonstrates that the shift of these Thy-1+ NK cells to Thy-1- NK cells is influenced by the thymus: NK cells developing in the total absence of the thymus--bone marrow (bm) cells from adult thymectomized (Tx) or nude (nu) mice injected into thymectomized recipients, (Tx) bm----(Tx) or nu bm----(Tx)--remain Thy-1+, while the presence of the thymus at some stage of the NK cell maturation--bone marrow cells injected into thymectomized recipients, bm----(Tx), or normal recipients reconstituted with bone marrow cells from thymectomized or nude mice, (Tx) bm----or nu bm----normal recipients--is sufficient for the development of normal ratio of Thy-1+ and Thy-1- NK cells.

The effects of cyclophosphamide (Cy) on the different cell populations participating in the cytotoxic T lymphocyte (CTL) response against haptenated (trinitrophenyl, TNP) syngeneic cells were studied. Pretreatment of responder cell donor mice with 150 mg/kg Cy decreased the cytotoxicity against TNP-modified syngeneic target cells almost to the background level. When TH cells were added to the culture the cytotoxicity increased significantly. Helper T cells were generated in vivo by priming the mice with TNP-modified syngeneic spleen cells or sensitizing the mice with a reactive hapten (TNCB). However, if the TH cell donor mice were treated with Cy before in vivo priming, the cytotoxicity reached the normal level, which indicated that TH precursors were not destroyed by Cy treatment and TH induction was even more effective after Cy. These data indicate that the decrease of the response by this Cy dose is not due to the sensitivity of CTL or TH precursors. Mice could be primed with male-specific (HY) antigen in spite of Cy pretreatment. However, Cy pretreatment caused a latent period of 2 weeks when effective CTL could not be generated in vitro, but after that the capacity for CTL generation was restored. These experiments confirm that pretreatment of responder cell donor mice with Cy does not destroy CTL or TH precursors, but rather affects their in vitro restimulation probably by destroying a short lived 'inducer' cell that is needed.

Mouse antibodies were prepared to human amniotic fluid fibronectin by means of somatic cell hybrids. Comparison of immunological reactivity of antibodies with amniotic and plasma fibronectin revealed that the two molecules had very similar patterns of antigenic determinants. Antibodies from one cell population, however, bound better to amniotic fluid fibronectin, indicating that there is a difference in the molecular structure of fibronectins isolated from the two sources.

We have investigated whether cytolytic cells specific for hapten-coupled syngeneic cells have the same public idiotype as serum antibodies against the same hapten, NP-aminocaproyl (NP-cap). Anti-NP-cap antibodies of C57BL mice possess a public idiotype that can be detected by antisera against the whole V region of the antibody molecule or against the VH domain. Neither reagent had an effect on the cytolytic response either when they were used to sensitize the effector cells for complement killing or when used at high concentrations in the culture medium during the secondary stimulation in vitro.

Several of the biological effects of bacterial lipopolysaccharide (LPS) can be induced by the lipid A part of the molecule. Here we show that in human peripheral blood monocytes, synthetic E. coli lipid A is as effective as the whole LPS molecule in inducing the production of interleukin 1 (IL-1) bioactivity which remains associated to the cells (i.e. IL-1 alpha). In contrast, LPS- but not lipid A-stimulated cells released the bioactive IL-1 produced into the culture supernatant (mainly IL-1 beta). Northern blotting analysis demonstrated, however, that LPS and lipid A are equally effective in inducing the accumulation of IL-1 alpha and IL-1 beta mRNA. These data support the hypothesis that induction of IL-1 biosynthesis and activation of the secretory mechanism for IL-1 are independent phenomena.

Mice were primed with the hapten 3-nitro-4-hydroxy-5 iodophenacetic acid (NIP) conjugated to chicken globulin (cg) and were boosted 2, 6, or 12 months later with CG conjugates of the related haptens 3,5-diiodo-4-hydroxyphenacetic acid (DIP) or 3-nitro-4-hydroxphenacetic acid (NP). Accelerated secondary responses were demonstrated both in the 7S and 19S class. Fine-specificities of secondary-response antibodies were studied by the hapten inhibition method of haptenated bacteriophage inactivation. 7S antibodies were found to have the fine-specificity of anti-NIP antibodies regardless of whether DIP or NP was the booster hapten ('original antigenic sin'). 19S antibodies had the fine-specificity of anti-DIP when DIP was the booster hapten. NP as the booster hapten resulted in 19S antibodies whose fine-specificity was intermediate between anti-NIP and anti-NP. A strong B-cell memory could thus be demonstrated in the 7S antibody response and a weak B-cell memory in the 19S antibody response.

Cells exerting a veto type of suppressive activity, that is, suppressing the cytotoxic T lymphocyte formation against the class I major histocompatibility complex antigen on their surface, can be found in different lymphoid tissues, for example, in the bone marrow. In this study we show that there is variation in the vitro veto-cell activity between bone marrow cells derived from different mouse strains: bone marrow cells derived from mice carrying the non-H-2 genes of B10/B6 mice had clearly the strongest veto activity.

Graft-versus-host disease (GVHD) was induced in (CBA X C57BL/6) F1 mice by i.v. injection of 50 X 10(6) parental spleen cells. The GVHD induced an enhanced NK (anti-YAC-1) cytotoxicity during the first 2 weeks after the spleen cell transfusion. This cytotoxic activity was shown to be mediated by asialo GM1-positive, partially Thy-1-positive and nylon-wool (NW) non-adherent cells, thus being classical NK cells. Depletion of NK-cell activity from donor and/or recipient mice with anti-asialo GM1 antibody prior to the spleen cell transfer did not prevent the GVHD as judged by the splenomegaly assay. Also, when NK activity was potentiated with polyinosinic-polycytidylic acid (pIC), no effect on the GVHD was seen. These data suggest that NK cells are not crucial for the development of GVHD in this model.

Production of IgA anti-hapten antibodies in vitro was studied in suspension and fragment cultures of spleens from mice immunized with NIP-CG (4-hydroxy-5-iodo-3-nitrophenylacetyl coupled to chicken globulin) a few weeks earlier. IgA anti-hapten production tended to be of much shorter duration than IgG anti-hapten production. IgA produced in fragment cultures had sedimentation coefficients of 9S, 11S, and 13S. 9S IgA anti-hapten was more prominent later during the in vitro response than earlier. In suspension cultures IgM anti-hapten production preceded IgA and IgG anti-hapten production. Very little IgM anti-hapten was seen in fragment cultures, although the responses on a cell-to-cell basis tended to be much higher in fragment than in suspension cultures.

We studied the role of HLA class II antigens in the regulation of interleukin 1 (IL-1) production in human monocytes. Monocytes were cultured with monoclonal anti-HLA-DR antibodies for 24 h after which cellular (i.e. intracellular and membrane-associated) IL-1 production, IL-1 secretion, and the expression of IL-1 alpha and IL-1 beta mRNA were determined. One of the anti-HLA-DR antibodies tested (anti-HLA-DR, Becton Dickinson) clearly induced IL-1 alpha and IL-1 beta mRNA expression and cellular IL-1 production. The other anti-HLA-DR antibody tested (OKIa1, Ortho) had no effect on IL-1 production. The stimulatory effect of anti-HLA-DR was enhanced by IFN-gamma in both fresh and aged monocytes. A synergistic effect by anti-HLA-DR and suboptimal doses of LPS (1 ng/ml) on both cellular IL-1 production and secretion was also demonstrated. The possibility of contaminating LPS causing the IL-1-inducing effect of anti-HLA-DR was excluded by the inability of polymyxin B to abolish the anti-HLA-DR-induced IL-1 production.

Both cortical and medullary thymocytes could give rise to both allospecific and hapten-specific cytotoxic T lymphocytes (CTLs) if the cultures contained the T-helper-cell-replacing hormone, interleukin 2 (IL-2). The H-2 restriction specificity of these CTLs was tested by using three different specificity determinants that are known to be influenced by the H-2 genotype of the thymus (rather than by the H-2 of the precursor cell itself): the cross-killing of trinitrophenyl (TNP) coupled allogeneic targets in anti-TNP self responses; the dominance of H-2k-restricted CTLs over H-2d-restricted CTLs in the response of (H-2k X H-2d)F1 mice to TNP-self; and the capacity of alloreactive CTLs to cross-reactively lyse TNP-coupled syngeneic cells. The experiments show that, with regard to all of these specificity determinants, CTLs derived from these intrathymic populations are similar to CTLs derived from the adult mouse spleen. These data suggest that the H-2 restriction specificity is dictated before or at the same time that the cell acquires the capacity to respond to allogenic or haptenated syngeneic cells.

We have studied the effect of interferon in vivo (induced by polyinosinic-polycytidylic acid (pIC] on the natural killer (NK) cell lytic activity and on the numbers of large granular lymphocytes (LGL) and target-binding cells (TBC) in different lymphoid compartments. One day after the pIC induction the spleen contained two- to four-fold increased lytic activity without a significant change in percentages of TBC and LGL. In the peripheral blood the lytic activity was 12- to 40-fold higher, and a concomitant clear increase in the number of LGL and TBC was seen. In the lungs the increase in lytic activity was 8- to 16-fold and in the number of LGLs ca. 3-fold. These results demonstrate that not only the lytic activity of the pre-existing NK cells but also the total amount of NK cells is clearly elevated in the body, but this could be seen more significantly in other highly NK-active organs than the spleen.

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).

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.

Proliferative T cell responses against syngeneic (SMLC) and allogeneic (alloMLC) non-T cells were investigated during the splenic regeneration after an injection of a sublethal dose (300 mg/kg) of cyclophosphamide (Cy). Two days after Cy injection spleen contained no cells capable of proliferative responses. T cells from the regenerating spleens (on day 6-8) had a defective proliferative capacity: the reactivity in the SMLC was at the normal level while alloreactivity was negative. The alloMLC appeared by 14 days after Cy injection, and it was still lower than the alloMLC of the normal splenic T cells. Responding cells of the SMLC in the regenerating phase belonged to the Ly-1+2- cell population: anti-Ly-1 antibody + complement (C) diminished the SMLC by 80%, anti-Ly-2 antibody + C had no effect on the responsiveness of the regenerating T cells. The stimulation of this SMLC was less dependent on splenic adherent cells than the normal SMLC. The significance of these data to the origin of alloreactivity and to the role of the SMLC in the cytotoxic T cell responses are discussed.

Hapten-protein conjugates stimulated very poor anti-hapten responses in mouse spleen fragment cultures from unimmunized mice, whereas hapten coupled to type III pneumococcal polysaccharide or polylysine induced good responses. When the donors of the fragments were primed with the carrier protein, hapten-protein conjugates induced a strong anti-hapten response. Both the true primary and the carrier-primed response in vitro consisted mainly of IgA antibodies of 9-13S. In carrier-primed responses also IgM was produced at the beginning and IgG at the end of those responses.

The requirement for cell division during the maturation of natural killer (NK) cells was studied by following the appearance of donor-type NK cells in irradiated mice injected with bone marrow cells and by blocking the cell division at different times during this development. Irradiation (700 rad) or treatment with hydroxyurea (1 mg/g body weight, twice daily) of the recipient mice 7 days after the bone marrow cell inoculation inhibited the appearance of normal NK cell levels, suggesting that the NK cell progenitors are dividing cells. Blocking of the cell division in chimeras that had already developed high NK levels decreased the splenic NK activity, indicating the presence of a dividing NK cell population at this stage of maturation. These results are in accordance with the concept that mature NK cells are nondividing cells but are derived from actively proliferating progenitors in the bone marrow, and some of the first NK cells appearing in the spleen from the bone marrow can still be dividing.

The effect of gamma interferon (IFN-gamma) on interleukin 1 (IL-1) production by human monocytes induced either by bacterial lipopolysaccharide (LPS) or silica dust was examined. Release of biologically active IL-1 from LPS-activated monocytes was enhanced in a dose-dependent manner with IFN-gamma. The augmenting effect of IFN-gamma was most marked in monocytes stimulated with a sub-optimal dose of LPS (1 ng/ml). IFN-gamma delivered the augmenting effect only if present at the beginning of the IL-1 induction. The IL-1 released in the presence of IFN-gamma was characterized by gel filtration on fast protein liquid chromatography (FPLC) and by isoelectric focusing; the augmented form of IL-1 was a 17 kDa molecule, with pI 7, i.e. IL-1 beta. In contrast to these data, IFN-gamma had a decreasing effect on IL-1 release by monocytes stimulated with silica dust; in monocyte cultures stimulated with a sub-optimal dose of silica (100 micrograms/ml), only minute amounts of biologically active IL-1 were released in the presence of IFN-gamma. With higher silica concentrations the decreasing effect was less strong. The effects of IFN-gamma on the levels of IL-1 beta mRNA were also analysed. IFN-gamma did not alter the accumulation of IL-1 beta mRNA in LPS-stimulated monocytes. In contrast, in silica-stimulated monocytes IFN-gamma reduced the steady-state levels of IL-1 beta mRNA. Thus, these data indicate that, depending on the stimulating agent, IFN-gamma either down- or up-regulates IL-1 production; the former takes place mainly at the level of transcription, while the latter occurs with post-transcriptional mechanisms.

By means of semisyngeneic bone marrow transplantation, the appearance of donor-type natural killer (NK) cells in different organs (spleen, blood, lungs) of the recipients was studied. Seven days after the transplantation the first donor-type NK cells appeared in all these organs, and adult NK levels were reached simultaneously within a couple of days. The first NK cells to appear in every organ divided rapidly (sensitive to hydroxyurea) and contained a high proportion of Thy-1+ cells. These data suggest that NK cell precursors mature locally and simultaneously in different organs.

In the present study we investigated the development of natural killer (NK) cell lytic activity, and its correlation with the appearance of cells with large granular lymphocyte (LGL) morphology after bone marrow transplantation (BMT). NK activity was first found 7 days after bone marrow (BM) reconstitution, simultaneously with the appearance of the first LGLs. The number of LGLs, as well as the lytic activity, increased until Day 16 after BM reconstitution, after which they started to decrease, reaching the normal values of controls in 30 days. These early appearing LGLs differed somewhat from mature-type LGLs; they were larger, blast-like cells (found in the lower density fractions of Percoll gradient) and had a basophilic cytoplasma, in contrast to a pale cytoplasm in mature LGLs, but they expressed the asialo GM 1 (AGM 1) antigen like normal NK cells. Those NK cells that appeared first also tended to be lytically more effective than their mature counterparts. Taken together, these data suggest that the correlation between LGL morphology and NK lytic activity also holds true during the development of NK cells from their non-lytic precursors in the bone marrow.