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BackgroundObesity is a heritable complex phenotype which 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 relates with mortality.ObjectivesWe aimed to assess the cross-sectional association of BMI with age acceleration in twins to limit confounding by genetics and shared environment.Methods and ResultsParticipants were from the Finnish Twin Cohort (FTC; n = 1424), including monozygotic (MZ) and dizygotic (DZ) twins, 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 1-unit BMI increase. Additionally, heavier twins in a BMI-discordant MZ twin pair (ΔBMI > 3 kg/m2) had an epigenetic age 5.2 months older than their lighter co-twin (p-value = 0.0074). We also found a positive association between log(HOMA-IR) and age acceleration, confirmed by a meta-analysis of the FTC and two other Finnish cohorts (overall effect = 0.45 years, p-value = 0.0025) from adjusted models.ConclusionWe 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.

Increased levels of circulating cell-free DNA (cf-DNA) are associated with and predict poor health outcomes. However, its predictive ability for mortality in population-based samples remains understudied. We analysed the capability of cf-DNA to predict all-cause mortality and assessed whether it adds predictive value on top of the other risk factors in the Health 2000 survey (n = 1,257, 46–76 years of age, 15-years-follow-up, 18% deceased). When analysed in a multivariate model with the other factors that independently predicted mortality in the sample (age, gender, self-rated health, smoking and plasma levels of glucose and adiponectin), increases in cf-DNA levels were associated with increased risk of mortality (hazard ratio [HR] for 0.1 µg increase in cf-DNA: 1.017, 95% confidence interval [CI] 1.008–1.026, p = 0.0003). Inclusion of cf-DNA in the model improved the model fit and discrimination. Stratifying the analysis by cardiovascular disease (CVD) status indicated that cf-DNA predicted mortality equally well in individuals with (HR 1.018, 95% CI 1.008–1.026, p = 0.002) and without (HR 1.018, 95% CI 1.001–1.035, p = 0.033) CVD. In conclusion, our study indicates that cf-DNA level predicts mortality in middle-aged and older individuals, also among those with established CVD, and adds significant value to mortality prediction. Our results thus underscore the role of cf-DNA as a viable marker of health.

Self-rated health (SRH) is one of the most frequently used indicators in health research. It is easy to use and considered as a valid measure of health because of its robust association with mortality. This association can largely be explained by the fact that in SRH summarized many health factors such as disease, functioning, and symptoms. Yet the biological basis of SRH is poorly understood. We investigated the association of SRH with several biomarkers measured from blood and urine in a large population sample where disease and functioning could be taken into account. Data were from MARK- AGE, a European-wide project on biomarkers of aging. The sample included 3252 individuals aged 18 to 92 years from eight European countries. SRH was categorized as poor&fair, good, very good and excellent. Multinomial logistic regression was used, contrasting “excellent” to “poor&fair”. Altogether 170 biomarkers were included in the preliminary analyses and, adjusted for age and gender, 42 of them showed a significant association with SRH. Adjusted for age, gender and cardiovascular diseases, arthritis, diabetes, elevated blood pressure, hypothyroidism/hyperthyroidism and respiratory diseases, still 33 biomarkers showed a significant association. Of these, the TOP5 strongest SRH-associations were with concentrations of beta-carotene (β=1.7), 25-hydroxyvitamin-D (β=0.03), lutein (β=3.7), beta-cryptoxanthine (β=2.3) and HDL-cholesterol (β=1.3) in plasma (all P

Objective. There is ample evidence demonstrating that accelerated atherosclerosis prevails in autoimmune rheumatic diseases, particularly in SLE, and that the risk is due not only to traditional cardiovascular risk factors but also to the disease itself. ANAs are a hallmark of SLE and are known even to antedate the development of SLE. Our aim was to investigate whether positive ANAs in young adults are associated with risk factors for atherosclerosis or subclinical markers of atherosclerosis. Methods. ANAs were examined by IIF using HEp-2 cells as substrate in 2278 participants in the Cardiovascular Risk in Young Finns Study for whom detailed data on cardiovascular risk factors and markers of subclinical atherosclerosis (including brachial flow-mediated dilatation, carotid compliance and carotid intima-media thickness) were available. Results. In multivariate analyses, adjusted for age, BMI, serum concentrations of CRP, triglycerides, high-density lipoprotein and low-density lipoprotein cholesterol, blood pressure and smoking habits, ANA positivity (titre > 160) was inversely associated (� ¼� 0.145; P ¼ 0.034) with carotid compliance in women. Conclusions. Our results indicate that ANA positivity is associated with decreased carotid elasticity in women, suggesting that mechanisms resulting in ANA production may be involved in the development of early atherosclerosis.

Fusion of immune spleen cells of mice immunized with CEA with a mouse myeloma cell line resulted in several hybrid cell lines secreting antibodies to CEA as tested by radio-immunoassay. Four of these were cloned by limiting dilution and large amounts of anti-CEA antibodies were produced by growing the anti-CEA-producing cell clones intraperitoneally in mice and collecting the ascites fluids formed. Ascites fluids containing antibodies with both high and low affinity were obtained. The antibody having the highest affinity was shown to be specific to CEA, whereas one antibody showed cross-reaction with the normal cross-reacting antigen (NCA). The establishment of a competitive-inhibition doubleantibody radio-immunoassad with high sensitivity and specificity using mouse monoclonal antibodies is described.

Growing evidence points to a role for inflammation in prostate carcinogenesis. The significance of C-reactive protein (CRP), an inflammatory and innate immunity molecule, has not been evaluated thoroughly in prostate cancer (PC). In this study of 739 Finnish patients with PC and 760 healthy men, we evaluated the associations of CRP genotypes and haplotypes with total PC risk and PC progression, using prostate-specific antigen (PSA) as a marker of metastatic disease. Although the haplotype frequencies were similar in patients and controls, an association between haplotype ACCCA and patients' PSA levels was found. The carriers more often had a high PSA than non-carriers (P=0.0002) and the SNP rs2794521 A-allele and rs1800947 C-allele carriers had a higher PSA than non-carriers (P=0.009 and P=0.0004, respectively). A trend for a younger age at diagnosis was found among the carriers of ACCCA (P=0.07) and the rs1800947 C-allele (P=0.06), as well as a trend for the latter to have more likely metastases (P=0.06), but not after Bonferroni correction (alpha=0.00208). This is the first study to suggest association between PSA and CRP variants in PC and, therefore, further studies are warranted. CRP alleles previously found to protect against increased CRP levels are now suggested to be associated with metastatic PC, indicated by elevated PSA.

BACKGROUND: Apolipoprotein E (apoE) polymorphism plays a central role in lipid metabolism, but has recently also been suggested to regulate inflammation, as judged by levels of serum C-reactive protein (CRP). OBJECTIVE: To establish whether polymorphism of the apoE genes affects susceptibility to primary Sjogren's syndrome (pSS), degree of inflammation or age of onset of pSS. METHODS: ApoE genotype distribution and allelic frequencies were analysed using PCR and the TaqMan system in 63 Finnish Caucasian patients with pSS and in 64 healthy controls matched for sex, ethnic origin and area of residence. The clinical and immunological data on the pSS patients were analysed in relation to the apoE genotypes. RESULTS: There was no difference between pSS patients and controls in apoE genotype and allelic frequencies. The apoE epsilon4 allele was significantly associated with early onset of pSS in the entire population and in female patients (Kaplan-Meier log rank test, P = 0.0407 and P = 0.0168, respectively). The average age (+/- S.D.) of onset of pSS in all apoE epsilon4 allele carriers was 46 +/- 12 and in other genotypes it was 53 +/- 10 yr (P = 0.031, t-test). ApoE polymorphism was not associated with signs of inflammation evaluated by such markers as concentration of plasma CRP, plasma interleukin-6, plasma TNF-alpha, immunoglobulin G and haemoglobin, or leucocyte count or ESR. CONCLUSIONS: ApoE polymorphism does not affect susceptibility to pSS or levels of plasma inflammatory indices in patients with pSS. However, a clear association prevails between apoE epsilon4 and early onset of pSS.

Objectives To determine whether plasma matrix metalloproteinase 9 (MMP-9) and MMP9 (-1562C-->T) polymorphism have an effect on the disease phenotype in primary Sjogren's syndrome (pSS). Methods Plasma MMP-9 concentrations and polymorphism of the MMP9 gene were analysed in 66 patients with pSS. These data were studied in relation to the clinical data of the patients. The genetic data of patients were compared with the data of 66 healthy subjects. Results Plasma MMP-9 was higher in patients with definite pSS than in patients with possible pSS. This association was principally caused by higher plasma MMP-9 in patients with a positive Schirmer test and keratoconjunctivitis sicca. pSS patients with purpura, SS-A autoantibodies and RF had significantly lower plasma MMP-9 than patients without these characteristics. The overall MMP9 (-1562C-->T) allele frequencies were similar in patients and control subjects. The frequency of the allele T was higher in patients without Raynaud's phenomenon than in the control group. Conclusions MMP9 (-1562C-->T) could not be used for risk assessment in pSS. The presence of the rarer allele T may decrease the risk of Raynaud's phenomenon in pSS. High plasma MMP-9 is indicative of definite pSS but may paradoxically have a preventive effect on the eruption of purpura and on the development of autoantibody reaction in pSS.

Background: The production of proinflammatory cytokines activates the systemic inflammatory response in sepsis. Patients also develop a compensatory anti-inflammatory reaction, which may have an important down-regulatory effect on the overactive inflammation. However, the role of this anti-inflammatory response in sepsis is not completely clarified. In this prospective study, we investigated the relationship between the pro- and anti-inflammatory cytokine profiles in severe sepsis and their role in the development of multiple organ failure (MOF). Methods: Thirty-eight patients meeting the criteria for severe sepsis were studied. MOF was defined as a maximum SOFA score of 10 or higher. Serial measurements of the proinflammatory IL-6 and IL-1β and the anti-inflammatory IL-10 and IL-1ra were used. The cytokine samples were taken at the onset of sepsis and on the third and fifth day during the ICU period. Results: The initial IL-10 and IL-1ra responses were identical in patients with or without MOF. The anti-inflammatory cytokine levels remained elevated in the MOF patients, whereas in patients without MOF the levels declined. The IL-6/IL-10 ratio was significantly higher in the MOF patients on days 1 and 3 compared with patients without MOF. Conclusions: We could not demonstrate overproduction of anti-inflammatory IL-10 in MOF patients. On the contrary, the high IL-6/IL-10 ratio indicates that IL-10 deficiency may contribute to the development of MOF in severe sepsis.

Objectives. To determine whether plasma interleukin-6 (IL-6) and G/C base exchange polymorphism at position -174 of the IL6 gene have an effect on the clinical manifestations of primary Sjogren's syndrome (pSS). Methods. Levels of circulating IL-6 protein and polymorphism of the IL6 gene were analysed in 66 patients with pSS and in 400 healthy subjects. These data were studied in relation to clinical data on the pSS patients. Results. Plasma IL-6 was elevated in pSS patients compared with healthy controls, pSS patients with coeliac disease, pulmonary fibrosis or alveolitis or peripheral nervous system symptoms had significantly higher IL-6 levels than patients without these manifestations. IL-6 levels increased in parallel with the histological grade of minor salivary gland biopsy and the number of pSS criteria fulfilled. IL6 allele frequencies were similar in patients and normal subjects. Plasma IL-6 levels were regulated by the IL6 genotype in pSS patients. Conclusions. The G/C polymorphism of the IL6 gene does not predispose patients to pSS, but the circulating IL-6 concentration is related to specific manifestations of the disease and the levels of IL-6 are regulated by the IL6 promoter polymorphism in pSS.

We tested the effects of interleukin-2 (IL-2), human recombinant tumor necrosis factor alpha (TNF-α), Staphylococcus aureus Cowan I (SAC), TPA, and their combinations, using a standard thymidine incorporation assay, in order to identify an optimal mitogen combination (OMC) for 24 consecutive patients with B-cell chronic lymphocytic leukemia (B-CLL). The combination that induced the highest thymidine incorporation was chosen as the OMC for each patient. Among 14 mitogen combinations tested, there were six different OMCs, of which the most frequent was TNF-α + IL-2. It was the OMC in 9 of 24 cases. The other OMCs were TNF-α + TPA1 (5/24), SAC + IL-2 (5/24), TPA1 + IL-2 (3/24), TPA10 + IL-2 (1/24), and TNF-α + TPA10 + IL-1 (1/24). The mitogenic power of the selected OMC in each case was then evaluated both by the combination of immunophenotyping and molecular cytogenetic techniques known as MAC (Morphology, Antibody, Chromosomes) and standard chromosome analysis. After OMC stimulation, the levels of DNA synthesis and B-cell proliferation (mitotic index) were, on average, 10-fold higher than those observed after standard TPA stimulation (P < 0.0001). The proportion of mitotic B cells exceeded the proportion of mitotic T cells in 70.1% of the cases after OMC stimulation. After TPA stimulation, 7.7% ± 2.5% of all mitoses were B-cell mitoses, whereas after OMC stimulation this proportion rose to 57.9% ± 5.3%. The frequency of clonal chromosomal aberrations increased from 46% after TPA stimulation to 79% after OMC stimulation. The clonal aberrations del(6q), del(11q), and/or del(13q) were observed in 26%, 32%, and 42% of the patients with the respective clonal chromosomal aberrations, whereas the corresponding frequencies after TPA stimulation were only 4%, 21%, and 17%. When the lineage involvement of cells with clonal chromosomal aberrations from three patients was analyzed, the aberrations were found to be restricted to B cells only, and in one patient to a minor subset of B cells. The results demonstrate that an individually chosen OMC induces a high rate of proliferation in neoplastic B cells. We found deletions in 6q, 11q, and 13q at higher frequencies than reported previously, most probably as a result of an improved mitogenic response. The identification of an optimal mitogen stimulation for each patient, prior to chromosome analysis, can well be expected to reduce the rate of false-normal results in the future. This is essential for accurate evaluation of the prognostic significance of chromosomal aberrations in B-CLL.

Summary Human cytomegalovirus (CMV) infection is associated with a higher risk of cardiovascular disease in immunocompromised organ transplant patients. It has been linked with the pathogenesis of elevated arterial blood pressure. However, controversy exists as to whether CMV infection is associated with endothelial function, and little is known about its role as a potential risk factor for early atherosclerosis development at a young age. We aimed to discover if CMV antibody titres are associated with early vascular changes (carotid intima-media thickness, carotid artery distensibility and brachial artery flow-mediated dilation), blood pressure elevation or other traditional cardiovascular risk factors. CMV antibody titres were measured in 1074 women and 857 men (aged 24–39 years) taking part in the Cardiovascular Risk in Young Finns study. CMV antibody titres were significantly higher in women compared to men. In men, high CMV antibody titres were associated directly with age (P

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative conditions characterised by the presence of abnormal accumulation of proteins and loss of neurones in specific regions of the brain. The aetiology and pathogenesis of AD and PD still remain largely unknown. Evidence has emerged that immune mediated mechanisms may be important in the development of these disorders,1,2 and cytokine interleukin 1 (IL1) is one of the mediators suggested to be involved. The IL1 family comprises three proteins, the proinflammatory IL1α and IL1β and their inhibitor IL1 receptor antagonist (IL1Ra), which are encoded by the IL1A , IL1B , and IL1RN genes respectively.3 Since IL1 may have a role in AD4–8 and in PD,9,10 variation in the IL1A , IL1B , and IL1RN genes may be of importance in the development of these disorders. There is already some evidence in support of this, including the postulated association of the IL1A (−889) \*2/\*2 genotype with AD11 and the influence of the IL1B (−511) \*1/\*1 genotype on age at onset of PD.12 However, validation in different population groups is needed to determine whether polymorphisms located in the IL1A , IL1B , and IL1RN genes represent actual susceptibility factors for AD and PD. In the present study, our purpose was to investigate whether polymorphisms of the IL1 cluster genes are associated with the risk of AD or PD in Finnish patients. Ethical approval for the investigation was obtained from the local Hospital Ethical Committees. All patients and controls enrolled in the study were from western Finland. The AD group consisted of 92 sporadic late onset (>65 years) patients (63 women, 29 men). Thirty-eight patients (mean age at onset 73.7 years (SD 4.9)) met the clinical diagnostic criteria of the NINCDS-ADRDA Work …

Activin A/erythroid differentiation factor (EDF), a dimeric polypeptide hormone composed of two beta A subunits, regulates growth and erythroid differentiation of human hematopoietic progenitor and erythroleukemia cells. We have identified activated human peripheral blood monocytes as a natural source of activin A/EDF. In these cells, lipopolysaccharide (LPS) induced rapidly the expression of the beta A subunit mRNAs through protein kinase C-dependent transcriptional regulation. The beta A subunit mRNA expression was also increased by 1,25-dihydroxyvitamin D3, an inducer of macrophage maturation of monocytes. Western analysis with an anti-beta A antibody and an erythroid differentiation bioassay confirmed that the conditioned media of LPS-activated monocytes contained the activin A/EDF protein. We suggest that monocyte/macrophage-derived activin A/EDF may not only modulate hematopoiesis but may also act as a mediator molecule in the diverse physiologic and pathogenetic events in which these cells are involved.

Although C-reactive protein (CRP) is known to predict cardiovascular events, its status as a causal risk factor is still controversial. CRP gene single nucleotide polymorphisms (SNPs) have been shown to associate with CRP concentration, but no direct independent effect on early atherosclerotic changes has been demonstrated. We aimed to determine if CRP gene polymorphisms or haplotypes are associated with CRP concentration or carotid artery compliance (CAC), an indicator of subclinical atherosclerosis. We genotyped CRP gene polymorphisms −717A>G, −286C>T>A, +1059G>C, +1444C>T and +1846G>A and measured CRP concentration and CAC in 2283 young adults participating in The Cardiovascular Risk in Young Finns Study. A strong association was found between CRP genotypes and CRP concentration, which was also seen at the haplotype level. Linear regression analysis showed an independent effect of each SNP on CRP concentration after adjustment for risk factors, except for +1444 in males. Moreover, −286C>T>A, +1444C>T and +1846G>A were associated with CAC in males, but not in females. Men carrying the SNP −286 allele C had increased CAC after adjusting for risk factors. These data suggest that the presence of high producer CRP genotype is deleterious to carotid elasticity in men.

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.

This study involved 342 patients hospitalized because of severe, persistent sciatica suggestive of a lumbar intervertebral disc herniation. After standard clinical evaluation. EMG and myelography, 220 patients underwent lumbar discectomy. The remaining 122 patients were treated conservatively. Follow-up examinations were arranged after 1, 5 and 13 years. The study focused on the rehabilitation outcome in general and differences in outcome between the two treatment groups. Several indicators showed a rather poor outcome for sciatica patients during the 13-year follow-up period. In the operated group 16% had been re-operated because of lumbar disc herniation. True recurrence of herniation (same level and side) occurred in 8%. In the conservatively treated group 14% had undergone spinal surgery. Nearly 70% of the patients still reported sciatica. Self-assessed levels of low back pain were “no change/worse” for 19% in the operated group and for 44% in the conservatively treated group. In both the study groups, nearly 40% of the subjects had retired on disability pensions.

Interleukin (IL)-1 beta and IL-6 levels in plasma and cerebrospinal fluid (CSF) of 14 medicated schizophrenic patients and 9 controls were measured by enzyme-linked immunosorbent assay. Almost no detectable levels of IL-6 in plasma or CSF from patients or controls were found. With regard to mean plasma or CSF levels of IL-1 beta there were no statistically significant differences between the two groups investigated. The dose of neuroleptic medication the patients used did not correlate with the IL-1 beta plasma levels. For the whole series of subjects (n = 23) or for the schizophrenic patients (n = 14) no correlation between the plasma and CSF levels of IL-1 beta was found.

The interleukin (IL)-1 gene cluster within chromosome 2 contains genes coding for both anti- and proinflammatory cytokines, including IL-1alpha, IL-1beta, IL-1Ra, and several novel cytokines designated IL-1F5 - IL-1F10.1 Proinflammatory cytokines IL-1alpha and beta are involved in the enhancement of inflammation and host defence. Anti-inflammatory IL-1Ra counteracts the function of IL-1alpha and IL-1beta. These cytokines are produced by a variety of cell types, for example, monocytes, magrophages, and keratinocytes. All IL-1 family genes are polymorphic and several of these polymorphisms have been shown to be associated with either susceptibility to or severity of inflammatory conditions and diseases.2 Atopy is a familial syndrome underlying atopic eczema, allergic rhinoconjuctivitis, and bronchial asthma. The skin prick test (SPT) is used to examine specific IgE mediated allergic responses and the results are generally in line with anamnestic data on atopy.3 Both IL-1alpha and beta have been shown to play a role in the regulation of allergen induced early and late reactions.4,5 IL-1alpha has also been found to accelerate cutaneous inflammation,6 whereas IL-1beta is involved in the regulation of contact sensitisation.7 In asthmatic bronchial epithelium, the expression of both IL-1beta and IL-1Ra is increased.8 Cytokines function as a network and thus the net effect on allergic inflammation may be dependent on allele combinations of individual cytokine genes. Recently in our population based case control study, we showed that IL1A (SNP +4845) genotype influences the SPT results.9 The carriers of the rarer allele 2 had a significantly lower SPT positive rate than that of non-carriers. Now we have expanded the study by analysing two additional SNPs ( IL1B SNP +3954 C>T and IL1RN VNTR in intron 2) in the IL-1 gene cluster from the same cohort. Altogether 254 females and 151 males (mean age 60 years, age range …

We read with interest the paper by Joos et al 1 on the association of IL-1 gene haplotypes with decline in lung function in smokers and share their view on a possible role of IL-1 genetics in inflammatory respiratory diseases. We have analysed the same polymorphism by the same methods in adult incident non-smoking asthmatic patients and non-smoking controls. Our results indicate that the association of IL-1 genetics with rate of decline in lung function is not limited to smokers. New adult asthma cases and controls were selected from a cohort of the Mini-Finland Health Survey (MFHS) and later re-evaluated. A more detailed description of the methods used in MFHS has been published elsewhere.2 The accuracy of the method of asthma case ascertainment has also recently been described.3 IL-1 haplotypes …

Cross-reactive T-cell cytotoxicity is seen when cytotoxic responses are generated in mixed lymphocyte cultures either between mouse strans which differ at the major histocompatibility complex, H-2, or between H-2b mutant strains and the strain from which they were derived. This cross-reactivity can be measured with [51Cr] labeled target cells from a number of different H-2 haplotypes, and the pattern of cross-reaction indicates that the target antigens are unlikely to be any of the serologically defined public specificities. In contrast, the specificity of H-2 restricted cytotoxic responses, such as that to the male-specific antigen, H-Y, is exquisite, and male cells from strains of mice carrying H-2 haplotypes other than the responder have never been found to act as appropriate targets. The contrast between the specificity of anti-H-2 and H-2 restricted responses may argue for a greater idiotypic homogeneity of the cells makiing H-2 restricted responses, and the greater specificity of these responses may be necessary for their biological function.

Cyclophosphamide (Cy), an alkylating agent, has widely been used as an immunosuppressive drug both in clinical medicine and experimental immunology. This agent itself has no immunosuppressive capacity, but it is activated by liver microsomal enzymes. We have studied its immunosuppressive effect using a mouse T-cell function, the cytotoxic response against hapten-modified syngeneic cells, as the model. As we have reported previously, this T-cell function is more sensitive to Cy than the cytotoxic response against allogeneic cells. Now we have found that the hapten-specific cytotoxicity in one mouse strain, CBA, is much more resistant to Cy than in other mouse strains; 5–10 times higher Cy amounts are needed in CBA than, e.g., in C57BL/6 mice to get a comparable suppressive effect. This CBA character was shown to be recessive and this trait was not coded for by the genes in the H-2 complex. The experiments where responder cells were pretreated in vitro with sera from either CBA or C57BL/6 mice injected previously with Cy demonstrated that this differential Cy sensitivity was caused by differences in the activation of Cy rather than by differences in the sensitivities of the responder cells to activated Cy.

We compared the advantages of monoclonal antibodies (produced by plasma cell-myeloma cell hybrid lines) and those of conventional antibodies in the radioimmunoassay of digoxin. It was found that antibodies produced by some hybrid cell lines (hybridomas) were highly specific for the digoxin structure; this way the cross-reactions to related structures (e.g. spironolactone) could be avoided. When the hybridoma lines were grown in ascites, the resulting fluid could have as high or higher titre than the serum of a hyperimmunized rabbit. The high titre, the specificity and the permanent growth of the hybridoma lines make them an optimal source of the specific antibody in clinical radioimmunoassays for the measurement of drug or hormone levels.

Mouse monoclonal antibodies to carcinoembryogenic antigen (CEA) obtained by the somatic cell hybridization technique of Köhler and Milstein were used in a modified enzyme bridge immunoperoxidase staining method. Both high and low affinity antibodies were tested and their staining properties compared with those of a commercial polyvalent rabbit antiserum. The staining pattern of neoplastic epithelial cells in all seven antibodies in samples of primary adenocarcinoma of the colon was similar, indicating that no gross differences were found in the exposure of the different antigenic determinants of CEA in formalin fixed tissue. The background staining of the monoclonal antibodies are negligible. It is concluded that monoclonal antibodies are superior to conventional antisera in immunoperoxidase staining of CEA.

To develop monoclonal reagents for antigenic analysis and serotyping of Campylobacter spp., hybridoma cell lines were produced by fusion of mouse myeloma cells and spleen cells from mice immunized with Formalin-treated Campylobacter jejuni organisms. An enzyme immunoassay was used for preliminary screening of the cell culture supernatants and ascites. Twenty-nine clones which reacted with the immunogen were obtained. Seven of these clones were positive in passive hemagglutination tests with sheep erythrocytes coated with boiled saline extract of whole bacteria; four of these reacted with the purified polysaccharide preparation and with the autoclaved saline extract, but not with lipopolysaccharide prepared from the immunogen strain. Two of the antipolysaccharide clones agglutinated live bacteria in slide tests. Four additional clones gave positive slide agglutination tests with live bacteria, but in tube testing no clones agglutinated Formalin-treated bacteria. No cross-reactions with unrelated bacteria were seen, but several clones reacted in the enzyme immunoassay with many of the 24 Campylobacter strains studied. The clone which gave the highest mean enzyme immunoassay values with Campylobacter coli and C. jejuni strains also reacted with Campylobacter fetus subsp. veneralis and C. fetus subsp. fetus strains. This clone also gave the highest enzyme immunoassay value with an acid glycine extract of the immunogen, which indicates the presence of common antigens in the extract. The results suggest that monoclonal antibodies may be used to devise serotyping schemes for Campylobacter spp.

Hybrid cell lines secreting monoclonal antibodies against pregnancy-specific β1-glycoprotein (SP1) were produced by fusion of a mouse myeloma cell line with spleen cells from BALB/c mice immunized with purified placental SP1. The clones were further grown intraperitoneally in mice, and the ascites fluids contained anti-SP1 antibodies in high titers. One of the monoclonal antibodies was coupled to cyanogen-bromide-activated Sepharose and used for affinity chromatography of SP1 using late pregnancy serum as starting material. The purification of SP1 by means of immunoadsorption utilizing monoclonal antibodies offers remarkable advantages compared with the purification procedures used so far.

Mice were injected with a sublethal dose of cyclophosphamide (Cy) (300 mg/kg), and the appearance of the capacity of the regenerating spleen to form cytotoxic T lymphocytes (CTL) in response against 2,4,6 trinitrophenyl-coupled syngeneic cells or against allogeneic cells was followed. It was found that 8 da after Cy injection, the spleen contained cells that could give rise to CTL, but only 2,4,6 trinitrophenyl-specific CTL responses could be obtained at this stage. A low alloresponse was first seen 2 wk after Cy injection.

A series of surgically treated patellar tendon lesions among athletes is presented. The material was collected during 5 years from three sports injury clinics and from two hospitals. During this period the authors treated about 150 cases of jumper's knee, of which 34 cases were treated by operation. The athletes were mostly volley ball players, jumpers or runners. The operation revealed a necrotic focus of the patellar tendon in 21 cases, the retinaculum was thick and adherent in 16 patients and an exostosis of the patellar insertion was seen in two cases. The necrotic areas were excised, the thick and adherent retinaculum was divided and the exostoses were excised and drilled. Surgical treatment of chronic patellar tendon pains may give good results in selected cases.

The concentration of free and conjugated diazepam, of its major demethylated metabolite, N-demethyldiazepam, and of its hydroxylated metabolites, N-methyloxazepam and oxazepam, were measured by a GLC-method in plasma, bile and urine following four nightly doses of diazepam 10 mg. Ten patients with a T-tube in the common bile duct after choledochotomy (Group I) were studied and 12 patients after cholecystectomy (Group II). Twelve hours after drug administration, the mean total concentration of diazepam in bile was 1/23 that in plasma. Similarly, during 9–10 h only low concentrations of diazepam were found in the urine, and in both urine and bile only the unconjugated drug was found. The principal metabolite of diazepam in plasma was N-demethyldiazepam. In bile an average of 77% of the total amount of N-demethyldiazepam was in the conjugated form, and its total concentration was half that in plasma. In urine N-demethyldiazepam was mainly in the conjugated form. No hydroxylated metabolites of diazepam were found in plasma. Oxazepam was the metabolite found in bile and urine in the next highest concentration after N-demethyldiazepam. In the urine it was mainly conjugated, but in bile only a mean of 35% was conjugated. Both in bile and urine, N-methyloxazepam was found only intermittently and in low concentration. Diazepam and all of its common metabolites were measured in human bile, and the concentrations found were too low to produce a clinically significant enterohepatic circulation.

Monoclonal antibodies directed against Uukuniemi virus glycoproteins G1 and G2 in combination with polyclonal antibodies against the nucleoprotein (N) were used to study the maturation of the virus in Golgi complexes of infected chicken embryo fibroblasts and BHK cells. Of 25 monoclonal antibodies obtained, 10 were shown to be G1 specific and 15 were shown to be G2 specific by immunoblotting and immunoprecipitation. In double-staining experiments, some of the monoclonal antibodies gave similar distributions of fluorescence as compared with the staining obtained from polyclonal rabbit anti-G1-G2 antibodies. Others, however, preferentially stained either the glycoproteins in the Golgi complex or those at the cell surface. This may indicate that the glycoproteins underwent conformational changes during their transport. Uukuniemi virus infection resulted in the vacuolization of the membranes of Golgi complexes where the maturation of the virus was taking place. Double-staining experiments with monoclonal antibodies which preferentially stained the Golgi-associated viral glycoproteins and with anti-N polyclonal rabbit antiserum showed a correlation between the progressive vacuolization of the Golgi complex and the accumulation of viral nucleoprotein in the Golgi region, suggesting that a morphological alteration of the Golgi complex may be a prerequisite for intracellular maturation of the virus. Treatment of Uukuniemi virus-infected cells with tunicamycin, a drug which inhibits N-linked glycosylation, resulted in the accumulation of both glycoproteins at an intracellular location, apparently representing the endoplasmic reticulum. Double-staining experiments showed a parallel accumulation of nucleoprotein at these sites, indicating that local accumulation of glycoproteins is required for nucleoprotein binding to intracellular membranes.

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 response. 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 primary 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 immuno-regulatory role of AsialoGM1+ murine NK cells in the induction of allogeneic cytotoxic T cell responses was studied in vivo and in vitro. Depletion of ASGM1+ cells from the (CBAxC57BL/6)F1 cells used for the foot-pad immunization of CBA mice greatly reduced the formation of allospecific CTLs. However, highly purified ASGM1+ cells were not efficient stimulators of alloCTLs in vivo by themselves. When the same genetic combination was used in alloCTL stimulation culture in vitro, ASGM1+ cells were seen to suppress the activation of the CTL response. The opposite roles of ASGM1+ cells in the alloCTL activation in vivo and in vitro were seen when both lymphoid cells (spleen cells or peripheral blood cells) and non-lymphoid cells (epidermal cells) were used as stimulators. The data presented in this paper suggest that during the alloCTL activation, ASGM1+ cells have an important enhancing role in vivo; but in vitro these cells suppress the antigen-presenting cells.

Spleen cells from cyclophosphamide-treated mice responded in vitro to allogeneic stimulator cells but not to TNP-coupled syngeneic spleen cells. Similarly, cells from female mice, primed in vivo with syngeneic male cells, could not respond in vitro to male spleen cell stimulation if the mice were pretreated with cyclophosphamide. These results suggest that the precursor cells for H-2-restricted cytotoxic responses belong to a different T-cell subpopulation than the precursor cells for allogeneic responses.

The capacity of mouse spleen fragments to mount an anti-NIP (4-hydroxy-5-iodo-3-nitrophenylacetyl) response in vitro was studied. The fragments came from unprimed, NIP-Ficoll (polymer of sucrose and epichlorhydrin) or NIP-CG (chicken globulin) primed mice. Unprimed spleen fragments from C57BL/6 mice gave a good anti-NIP response to NIP-Ficoll, whereas CBA fragments did not. Priming with NIP-Ficoll made CBA fragments responsive and enhanced slightly the response of C57BL/6 fragments when stimulated with the same antigen. This memory effect could be seen only after a small priming dose. Priming the mice with NIP-Ficoll made their spleen fragments responsive to a protein conjugate of NIP (NIP-CG), but this effect was seen only after priming with a high dose. The antibody class distribution and the kinetics of the appearance of different immunoglobulin classes were similar in the primary and secondary responses in vitro. The peak responses of IgM, IgA and IgG were reached on day eight and the relative amount of IgG was the same in the primary and in the secondary responses. Spleen fragments derived from NIP-CG primed mice produced more IgG anti-NIP antibodies than fragments derived from untreated mice when immunized in vitro with NIP-Ficoll. The amount of IgG was, however, much higher when these fragments were challenged with the homologous antigen, NIP-CG.

Two cases of traumatic bilateral Achilles tendon rupture are reported. One of the patients was a healthy middle-aged man, who had been an active national-level gymnast 20 years earlier. He had not suffered any complaints of Achilles tendons before. The ruptures occurred when, after a sauna, he showed his guests a vault forwards, which he had been able to perform easily. This time the landing took place on the toes, causing a high peak stretch to the calf muscles and Achilles tendons. The total rupture of both Achilles tendons was treated surgically, with an excellent result 2 days after the trauma. End-to-end suturation and a fascial flap plasty were made on both sides. No macroscopic degeneration could be detected on the rupture sites. He was allowed to walk freely 6 weeks after the surgery. The second case was a 54-year-old woman, who had suffered from Achilles tendinitis and peritendinitis for 2 years. Both tendons had been surgically treated, and severe adhesions and local degenerative changes had been found. The tendon rupture occurred when she injured her left ankle while getting out of the car. Two days later she fell at home, because of the weakness of the left side, and consequently the right Achilles tendon was injured. She was treated conservatively for 10 days, before the surgery was performed. Both tendons were ruptured and an extensive degeneration of the area was observed. The right side suffered from a rerupture, which was again treated surgically. After surgery the recovery was slow, but the final result 3 years later was moderate. Neither of the patients had any systemic diseases.

A redox steady state is important in maintaining vital cellular functions and is therefore homeostatically controlled by a number of antioxidative agents, the most important of which are enzymes. Oxidative Stress (OS) is associated with (or/and caused by) excessive production of damaging reactive oxygen and/or nitrogen species (ROS, RNS), which play a role in many pathologies. Because OS is a risk factor for many diseases, much effort (and money) is devoted to early diagnosis and treatment of OS. The desired benefit of the “identify (OS) and treat (by low molecular weight antioxidants, LMWA)” approach is to enable selective treatment of patients under OS. The present work aims at gaining understanding of the benefit of the antioxidants based on interrelationship between the concentration of different OS biomarkers and LMWA. Both the concentrations of a variety of biomarkers and of LMWA were previously determined and some analyses have been published by the MARK-AGE team. For the sake of simplicity, we assume that the concentration of an OS biomarker is a linear function of the concentration of a LMWA (if the association is due to causal relationship). A negative slope of this dependence (and sign of the correlation coefficient) can be intuitively expected for an antioxidant, a positive slope indicates that the LMWA is pro-oxidative, whereas extrapolation of the OS biomarker to [LMWA] = 0 is an approximation of the concentration of the OS biomarker in the absence of the LMWA. Using this strategy, we studied the effects of 12 LMWA (including tocopherols, carotenoids and ascorbic acid) on the OS status, as observed with 8 biomarkers of oxidative damage (including malondialdehyde, protein carbonyls, 3-nitrotyrosine). The results of this communication show that in a cross-sectional study the LMWA contribute little to the redox state and that different “antioxidants” are very different, so that single LMWA treatment of OS is not scientifically justified assuming our simple model. In view of the difficulty of quantitating the OS and the very different effects of various LMWA, the use of the “identify and treat” approach is questionable.