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1.  Influence of smoking and race on immunoglobulin G subclass concentrations in early-onset periodontitis patients. 
Infection and Immunity  1996;64(7):2500-2505.
Recent data indicate that smoking is an important risk factor for the development of periodontitis. Smoking is also known to reduce serum immunoglobulin G (IgG) levels. Interestingly, patients with the localized form of early-onset periodontitis (LJP) have elevated levels of serum IgG2, and those who smoke are not clinically different from nonsmoking LJ subjects. In contrast, patients with the generalized form of early-onset periodontitis (G-EOP) who smoke have more extensive destruction than their nonsmoking counterparts. Given the effects of smoking on EOP and the association of IgG2 with less severe disease, we hypothesized that smoking might reduce serum IgG2 and that this might be most apparent in G-EOP. We therefore examined the effects of smoking on serum IgG subclass concentrations in race-matched groups: LJP, G-EOP, and age-matched periodontally healthy controls (NPs). Smoking status was established from serum cotinine levels, and serum IgG subclass concentrations were determined by using radial immunodiffusion. The data indicated that the effects of smoking were remarkably selective with respect to both IgG subclass and race. Smoking did not appear to have any effect on the concentration of IgG1 or IgG3 in either black or white subjects. In contrast, smoking was associated with depressed serum IgG2 concentrations in both white NP and G-EOP subgroups. Serum IgG2 levels in black subjects did not appear to be depressed by smoking, with the single striking exception of the black G-EOP subgroup which also had depressed serum IgG4 levels. The results here confirm that smoking has effects on serum immunoglobulin levels, but the effects were both race and serum IgG subclass specific. Furthermore, the periodontal diagnosis of EOP subjects appeared to be important, as indicated by the fact that IgG2 and IgG4 levels were reduced in smoking black G-EOP subjects whereas the IgG2 and IgG4 levels in black LJP and NP subjects were not reduced by smoking.
PMCID: PMC174103  PMID: 8698472
2.  Immunoglobulin allotypes and immunoglobulin G subclass responses to Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis in early-onset periodontitis. 
Infection and Immunity  1996;64(10):4226-4230.
The present study was performed to estimate the observed frequencies of the immunoglobulin heavy-chain (Gm) and light-chain (Km) allotypes among patients with early-onset periodontitis (EOP) and their effect on the IgG2 subclass responses against Actinobacillus actinomycetemcomitans Y4 and Porphyromonas gingivalis 381, respectively. Sixty-nine EOP patients, including 11 with localized juvenile periodontitis (LJP), 19 who had LJP, 15 with LJP-rapidly progressing periodontitis (RPP), and 24 with RPP, were examined for the Gm and Km allotypes by a hemagglutination inhibition test. Levels of immunoglobulin G2 (IgG2) antibodies against the two organisms were determined by enzyme-linked immunosorbent assay. Fifty race- and age-matched, periodontally healthy subjects were also included as a control group. The observed frequencies of the Gm haplotype afnb and Km(1) were significantly higher in the RPP and LJP groups, respectively. The G2m(n)+ group of those with RPP and the Km(1)+ group of those with LJP had significantly higher levels of IgG2 antibodies to A. actinomycetemcomitans and P. gingivalis, respectively. The results indicate that linkage disequilibrium of the G2m(n) locus in RPP patients or the Km(1) locus in LJP patients may be associated with high IgG2 antibody responses to the respective bacteria. It was reasoned that the IgG2 antibody responses are associated with the immunoglobulin allotypes. The function of IgG2 antibodies in their reaction to different bacterial antigens may be interpreted as either protective or nonprotective in the two different types of EOP (i.e., LJP and RPP).
PMCID: PMC174360  PMID: 8926092
3.  Influence of anti-Actinobacillus actinomycetemcomitans Y4 (serotype b) lipopolysaccharide on severity of generalized early-onset periodontitis. 
Infection and Immunity  1996;64(9):3908-3910.
The objective of this study was to determine if a relationship exists between antibody reactive with the Actinobacillus actinomycetemcomitans serotype b lipopolysaccharide (LPS) and severity of periodontal disease in generalized early-onset periodontitis (G-EOP). The concentration of antibody reactive with A. actinomycetemcomitans serotype b LPS was determined for 102 G-EOP subjects. Analysis of the relationship between antibody reactive with A. actinomycetemcomitans serotype b LPS and measures of periodontal attachment loss indicated that the patients with the highest concentrations of antibody reactive with A. actinomycetemcomitans serotype b LPS had significantly less attachment loss. These high-responder subjects also had anti-A. actinomycetemcomitans serotype b LPS with significantly higher relative avidity. The results suggest that antibody reactive with A. actinomycetemcomitans serotype b LPS is protective in G-EOP patients.
PMCID: PMC174312  PMID: 8751948
4.  Dendritic Cells Stimulated with Actinobacillus actinomycetemcomitans Elicit Rapid Gamma Interferon Responses by Natural Killer Cells  
Infection and Immunity  2004;72(9):5089-5096.
Human immunoglobulin G2 (IgG2) responses are gamma interferon (IFN-γ) dependent, and monocyte-derived dendritic cells (mDCs) promote IgG2 production. DCs spontaneously emerge from monocytes in cultures prepared from localized aggressive periodontitis (LagP) patients, and these patients have high levels of IgG2 that is reactive with Actinobacillus actinomycetemcomitans. These results prompted the hypothesis that an interaction between mDCs and A. actinomycetemcomitans promotes IFN-γ production, and IFN-γ is known to promote both immunopathology and protective IgG2. A. actinomycetemcomitans induced mDCs to produce interleukin-12 (IL-12), and the addition of A. actinomycetemcomitans and DCs to cultured peripheral blood lymphocytes elicited high levels of IFN-γ within just 24 h. In contrast, IL-4 was not detectable although DC-derived IL-10 production was apparent. A. actinomycetemcomitans-stimulated macrophages prepared from the same monocytes lacked the ability to induce IL-12 or IFN-γ responses. NK cells of the innate immune system were the primary source of this early IFN-γ, although CD8 T cells also contributed some. The NK cell-derived IFN-γ was IL-12 dependent, and A. actinomycetemcomitans-DC interactions were Toll-like receptor 4 dependent. A. actinomycetemcomitans and A. actinomycetemcomitans lipopolysaccharide (LPS) were more potent than Escherichia coli and E. coli LPS in the ability to induce DC IL-12 and IFN-γ. The ability of A. actinomycetemcomitans-stimulated DCs to induce NK cells to rapidly produce IFN-γ in the absence of detectable IL-4 suggests their potential for skewing responses toward Th1. This may help explain the presence of Th1-associated cytokines in gingival crevicular fluid (GCF) from LagP patients and the high levels of IgG2 in their serum and GCF that is reactive with A. actinomycetemcomitans.
PMCID: PMC517417  PMID: 15322002
5.  Blocking Pro-Inflammatory Cytokine Release Modulates Peripheral Blood Mononuclear Cell Response to Porphyromonas Gingivalis 
Journal of periodontology  2012;84(9):1337-1345.
Chronic periodontitis is an inflammatory disease in which cytokines play a major role in the progression of disease. Anti-inflammatory cytokines (IL-4 and IL-10) were reported to be absent or reduced in diseased periodontal tissues, suggesting an imbalance between the pro- and anti-inflammatory mediators. We have tested the hypothesis that there is cellular cross-talk mediated by pro- and anti-inflammatory cytokines and that blocking pro-inflammatory cytokine (TNF-α and IL-1) production will enhance anti-inflammatory cytokine (IL-4 and IL-10) production from peripheral blood mononuclear cells (PBMC) in response to P. gingivalis.
PBMC were isolated from individuals diagnosed with chronic periodontitis or healthy individuals and cultured for 24 hours. Concanavalin-A (ConA) was used as an activator of lymphocyte function. Live and heat-killed P .gingivalis or lipopolysaccharide from P. gingivalis was used as the bacterial stimulants. TNF-α and IL-1 production was neutralized by specific antibodies against TNF-α and IL-1α or β. Culture supernatants were evaluated by ELISA for TNF-α, IL-1β, IL-4, and IL-10 production.
Live P. gingivalis did not result in any significant IL-10 or IL-4 release while heat-killed P. gingivalis led to a significant increase in IL-10 levels compared to unstimulated or live P. gingivalis-stimulated cells from both healthy and periodontitis individuals. Overall, PBMC from patients with chronic periodontitis produced significantly lower IL-10 in response to ConA and P. gingivalis suggesting chronic suppression of the anti-inflammatory cytokine production. Blocking the pro-inflammatory cytokine response did not result in any substantial change in IL-10 or IL-4 response to live P. gingivalis. Blocking the pro-inflammatory cytokine response restored IL-10 production by cells from chronic periodontitis in response to P. gingivalis LPS.
These findings suggest that PBMC from patients with chronic periodontitis have suppressed anti-inflammatory cytokine production that can, in part, be restored by neutralizing pro-inflammatory cytokines. Monocytes are an important source of IL-10 production and monocyte-derived IL-10 might play a regulatory role in the pathogenesis of chronic periodontitis.
PMCID: PMC3935330  PMID: 23173823
IL-4; IL-10; monocytes; Porphyromonas gingivalis; Periodontitis
6.  Exercise on Prescription: trial protocol and evaluation of outcomes 
In many countries exercise prescriptions are used in an attempt to initiate a physically active lifestyle in sedentary populations. Previous studies have primarily evaluated low intensive exercise prescription interventions and found moderately positive effects on physical activity and aerobic fitness. In a highly intensive Danish exercise prescription scheme called 'Exercise on Prescription' (EoP) the general practitioners can prescribe EoP to sedentary patients with lifestyle diseases. The aim of this randomized trial is to assess the short- and long-term effects of the EoP scheme. Thus, the aim of this paper is to describe the randomized controlled trial designed for evaluating effectiveness of EoP, and to present results from validations of outcome measures.
EoP involves a 16-week supervised training intervention and five counselling sessions (health profiles). All patients referred to EoP were eligible for the trial and were offered participation during the baseline health profile. Comparisons between the EoP group and the control group were made at baseline, and after four and ten months. Physiological measures used were maximal oxygen uptake (VO2max), glycosylated haemoglobin (HbA1c), bodyweight, and BMI. Patient-reported measures used were physical activity, health-related quality of life, amount and intensity of exercise, compliance with national guidelines for physical activity, and physical fitness. The validation of the cycle ergometer test found a strong correlation between maximal work capacity and VO2max, and acceptable test-retest reliability at group level. Calibration of the HbA1c apparatus was stable over ten weeks with minimal use, and test-retest reliability was good. High agreement percents were found for test-retest reliability for the self-administered questionnaire.
The trial is designed to provide information about the effectiveness of the EoP scheme. The trial is part of a health technology assessment of EoP, which besides the effectiveness covers the patient perspective, the organization, and the health economy. All three methods validated were found useful for the EoP trial.
PMCID: PMC1820778  PMID: 17331263
7.  Serodiagnosis of Porphyromonas gingivalis infection by immunoblot analysis with recombinant collagenase. 
Journal of Clinical Microbiology  1996;34(10):2411-2413.
The Porphyromonas gingivalis collagenase-specific serum immunoglobulin A (IgA), IgM, and IgG responses from 20 patients with early-onset periodontitis (EOP), 20 patients with adult periodontitis, (AP), and 20 age- and sex-matched healthy controls were examined by immunoblot analysis. A recombinant collagenase antigen used for the immunoblot analysis was produced by using the plasmid pGEX-2T, which allows the fusion between the collagenase and glutathione S-transferase. There was no significant difference in collagenase-specific IgG antibody detection between samples from the EOP, AP, and control groups. In contrast, 85% of AP and EOP sera had collagenase-specific IgA antibodies, whereas only 20% of control sera showed collagenase-specific IgA reactivity. Plaque samples from all groups were assessed by PCR with primers complementary to the collagenase-encoding gene prtC. The results indicated that 90% of AP and EOP plaque samples and 10% of control samples were positive for P. gingivalis. All patients with collagenase-specific IgA antibodies were PCR positive. The results of the study indicate a nearly complete concordance (k = 0.856) between the presence of collagenase-specific IgA antibodies and PCR detection of P. gingivalis. By using PCR as the "gold standard," the sensitivity and specificity of the IgA immunoblot test were 94.7 and 90.9%, respectively. Therefore, the recombinant collagenase is a potential candidate for use in the serodiagnosis of periodontitis.
PMCID: PMC229282  PMID: 8880490
8.  Clinical and laboratory manifestations of elderly onset psoriatic arthritis: a comparison with younger onset disease 
Annals of the Rheumatic Diseases  1999;58(4):226-229.
OBJECTIVE—Although the influence of age on clinical and laboratory features has been widely demonstrated in many arthropathies, studies on elderly onset (> 60 years) psoriatic arthritis (EOPsA) are rare. This study compares manifestations at onset and two year outcome of EOPsA with those of younger onset PsA (YOPsA).
PATIENTS AND METHODS—Sixty six consecutive PsA patients with disease duration < 1 year, 16 EOPsA (>60 years) and 50 YOPsA (⩽60 years) were admitted to a prospective study. Clinical, laboratory, and radiographic assessment were carried out at admission and after two years. HLA class I and bone scintigraphy were also recorded. In 10 patients with EOPsA and 24 with YOPsA it was possible to obtain synovial fluid, which was subsequently analysed for local inflammatory indices, including interleukin (IL) 1β, IL6, and IL8.
RESULTS—Presenting manifestations of EOPsA differed from YOPsA in number of active joints (mean (SD)) (12.2 (6.3) v 6.7 (4.6), p<0.001), foot bone erosions (2.7 (1.2) v 1.1 (1.1), p<0.001), erythrocyte sedimentation rate (64.2 (35.3) v 30.5 (30.0) mm 1st h, p<0.001), C reactive protein (3.9 (2.0) v 1.3 (1.3) mg/dl, p<0.001) and synovial fluid IL1β (8.0 (4.7) v 3.0 (3.0) pg/ml, p<0.001) and IL6 (828.2 (492.6) v 469.3 (201.4) pg/ml, p<0.005). No differences were found in the number of subjects with dactylitis, pitting oedema, HLA-B27, or signs of sacroiliac and sternoclavicular joint involvement at bone scintigraphy. After two years, progression was more evident in EOPsA than in YOPsA, as the number of new erosions in the hands and also the C reactive protein were higher in EOPsA patients.
CONCLUSION—PsA has a more severe onset and a more destructive outcome in elderly people (onset >60 years) than in younger subjects. This behaviour may be influenced by immune changes associated with aging, as suggested by the higher concentrations of IL1β and IL6 found in the synovial fluid of EOPsA than in YOPsA.

 Keywords: elderly onset arthritis; psoriatic arthritis; synovial fluid; interleukins
PMCID: PMC1752862  PMID: 10364901
9.  Pregnancy and early onset pauciarticular juvenile chronic arthritis 
Annals of the Rheumatic Diseases  1999;58(8):475-480.
OBJECTIVES—To study interaction of early onset pauciarticular juvenile chronic arthritis (EOP-JCA) and pregnancy in the Polish population, in particular to confirm the ameliorating effect of pregnancy on disease activity reported by others and to analyse the factors that govern the occurrence of postpartum flare, with emphasis on the potential role of breast feeding.
METHODS—The reproductive outcome and disease status in 39 adult women with history of EOP- JCA was examined by means of a questionnaire and an interview. In all patients the disease onset occurred before the 6th birthday, 19 had persistent pauciarticular JCA (PeEOP-JCA) and 20 had extended pauciarticular JCA (ExEOP-JCA).
RESULTS—23 women had at least one successful pregnancy, seven had unsuccessful pregnancies but all of them had also one or more successful pregnancies. Among those who have never been pregnant (n=16) there was a higher frequency of eye disease and ExEOP-JCA compared with the rest of the group. In almost all cases pregnancy was associated with remission of disease activity, however a postpartum flare appeared after 22 pregnancies (52%). The flares were more frequent in women who had an active disease before pregnancy, had a flare after a previous pregnancy and/or were breast feeding.
CONCLUSIONS—In EOP-JCA patients pregnancy generally has a good outcome and induces amelioration of disease activity. After delivery, however, a flare of disease often appears, especially in women who were breast feeding, had a postparum flare previously or had an active disease before pregnancy. The pattern of interaction between disease and pregnancy found in EOP-JCA makes EOP-JCA similar in this respect to RA, but different from systemic lupus erythematosus and ankylosing spondylitis.

PMCID: PMC1752930  PMID: 10419865
10.  Heterogeneity of antibodies reactive with the dominant antigen of Actinobacillus actinomycetemcomitans. 
Infection and Immunity  1997;65(9):3794-3798.
The serotype b-specific carbohydrate antigen (SbAg) of Actinobacillus actinomycetemcomitans Y4 is reported to be the O antigen of lipopolysaccharide, and the highest titers of serum antibody reactive with A. actinomycetemcomitans in early-onset periodontitis (EOP) patients bind SbAg. These high titers of serum antibody reactive with SbAg are associated with a lesser extent and severity of periodontal disease. The aim of this study was to determine if a limited number of genes code for anti-SbAg antibodies as has been shown for immunoglobulin G (IgG) reactive with the type b polysaccharide from Haemophilus influenzae. Serum IgG reactive with the SbAg was prepared from 20 high-titer EOP patients by affinity chromatography. The IgG subclass concentrations were determined, and heterogeneity was analyzed by isoelectric focusing (IEF). IgG2 was the dominant subclass (83% of total IgG) in the anti-SbAg IgG fraction and represented an average of 1.33% of total serum IgG2. The IgG2 reactive with SbAg was isolated from the affinity-purified IgG fraction by affinity chromatography with protein A and subclass-specific monoclonal antibodies. On IEF gels, only 4 to 20 bands were observed in the anti-SbAg IgG fractions, indicating limited heterogeneity. N-terminal amino acid sequence analysis of eight representative anti-SbAg IgG2 preparations indicated that variable heavy and light chains consisted largely of V(H)III and V(kappa)II, respectively. However, a significant fraction of anti-SbAg may use V(H) and V(lambda) genes with blocked N termini. In short, these findings indicate that IgG reactive with SbAg is very much like the antibody reactive with H. influenzae type b polysaccharide. Similarities include IgG2 dominance, limited bands on IEF gels, supporting an oligoclonal response, and use of genes from V(H)III and V(kappa)II regions.
PMCID: PMC175541  PMID: 9284154
11.  Circulating promyelocytes and low levels of CD16 expression on polymorphonuclear leukocytes accompany early-onset periodontitis. 
Infection and Immunity  1997;65(9):3906-3912.
Early-onset periodontitis (EOP) is characterized by rapidly progressive alveolar bone loss, chemotactic defects of neutrophils, and significant familial aggregation. We found immature myeloid lineage cells, defined as promyelocytes, in the peripheral blood in patients with EOP. A hematological examination of peripheral blood cells showed normal reference values regarding cell proportions. Flow cytometry revealed significantly lower expression of CD16, a glycosylphosphatidylinositol (GPI)-anchored protein, on peripheral neutrophils in patients compared with those in age- and sex-matched healthy controls, whereas the levels of CD11a and CD11b expression were similar. The chemotactic response of neutrophils was lower toward not only formyl-methionyl-leucyl-phenylalanine but also complement fragment C5a than that of healthy controls. The expression of another GPI-anchored protein, CD14, was equally expressed by controls and patients. Therefore, the low level of CD16 expression was not due to the incomplete synthesis of the GPI anchor. GPI anchors of CD16 on neutrophils from controls and patients were both partially resistant to phosphatidylinositol-specific phospholipase C. The presence of promyelocytes in peripheral blood, low expression of CD16, and low chemotactic response of neutrophils suggest that patients with EOP have an abnormal maturation system in myeloid lineage cells in the bone marrow, which may be associated with the onset and course of EOP.
PMCID: PMC175557  PMID: 9284170
12.  Detection of altered T helper 1 and T helper 2 cytokine production by peripheral blood mononuclear cells in patients with multiple sclerosis utilizing intracellular cytokine detection by flow cytometry and surface marker analysis. 
Production of T helper 1 and T helper 2 cytokines was investigated in peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients by a newly described technique, detection of intracellular cytokines by flow cytometry in conjunction with immunophenotype analysis. T-cell gamma interferon (IFN-gamma) production and interleukin 10 (IL-10) production were examined after PBMC activation with T-cell mitogens at 5 and 24 h, and monocyte spontaneous production of IL-10 and production after PBMC activation with lipopolysaccharide (LPS) for 24 h were also examined. The data indicate that MS patients have decreased percentages of T cells capable of secreting IFN-gama compared with healthy controls, and this change is detectable at 5 and 24 h. the patients displaying decreased T-cell production of IFN-gamma were essentially confined to a group being treated with the newly approved drug Betaseron (berlex Labs, Cedar Knolls, N.J.), a recombinant form of IFN-beta (rIFN-beta 1b). By gating of the entire lymphocyte population, analysis of IFN-gama production in T cells (CD3+ versus that in non-T cells (CD3+) was possible. The percentage of IFN-gamma-producing lymphocytes that was made up of T cells was essentially unchanged between the Betaseron-treated patients, non-Betaseron-treated patients, and controls, indicating that the suppression of IFN-gamma production displayed by betaseron-treated MS patients was a nonspecific suppression of all IFN-gamma-producing lymphocytes as opposed to a suppression of T-cell production only. The data seem to indicate that treatment of MS with Betaseron corresponds to an inhibition of the lymphocyte's ability to produce IFN-gamma. No changes were detected in T-cell production of IL-10 at either time point. We also observed that MS patients in general appear to have small percentages of peripheral blood monocytes spontaneously producing slight but detectable levels of IL-10. No difference was seen regarding monocyte production of IL-10 after PBMC activation with LPS between MS patients and controls. Both populations responded with high percentages of monocytes producing IL-10. The data seem to indicate that treatment of MS with Betaseron, known to decrease the exacerbation rate of relapsing-remitting MS, corresponds to a suppression of peripheral blood lymphocyte production of IFN-gamma. Monocyte production of IL-10 may also play a role in regulating the disease process.
PMCID: PMC170359  PMID: 8807205
13.  Combined effects of in vitro penicillin and sickle cell disease sera on normal lymphocyte functions. 
Previously published work has shown that sera from healthy sickle cell disease (SCD) patients inhibits normal lymphocyte response to phytohemagglutinin (PHA) in vitro. The objective of the current study is to ascertain what the combined effects of SCD sera plus penicillin have on normal lymphocyte cytokine production and mitogenic response to PHA. Steady state sera from 20 SCD patients not on penicillin prophylaxis and 20 comparable healthy controls were used in all experiments. Four normal healthy individuals were used as donors for obtaining peripheral blood mononuclear cells (PBMC), by density gradient. PBMC with or without penicillin were PHA stimulated by standard in vitro culture for mitogenic response and cytokine production. Supernatant cytokine levels for interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)2 were quantified by ELISA technique. Results revealed suppression of mitogenic response in the SCD group with or without penicillin, compared to control sera (P < .001). Cytokine production in the SCD sera group showed increased production of IFN-gamma and TNF-alpha in the absence of penicillin, but suppression at all doses of penicillin. The control group results were as follows: no significant difference in IFN-gamma production with or without penicillin, mean TNF-alpha levels were the opposite of SCD sera with lower levels in the absence of penicillin. IL-2 production demonstrated a similar pattern for both groups of sera. IL-2 production was low without penicillin, but there was increased production with penicillin, which appeared dose related. The data suggests that sera from healthy SCD patients and in vitro added penicillin may have a combined suppressive effect on normal lymphocyte in vitro production of IFN-gamma and TNF-alpha. The current study results suggest that penicillin has the beneficial effect of decreasing TNF-alpha production and increasing IL-2 production when combined with SCD steady state sera. However, this in vitro benefit must be weighed against suppression of IFN-gamma production and ultimately, perhaps the long-term utility of penicillin prophylaxis in patients with SCD.
PMCID: PMC2594265  PMID: 12152923
14.  Novel Hairpin-Shaped Primer Assay To Study the Association of the −44 Single-Nucleotide Polymorphism of the DEFB1 Gene with Early-Onset Periodontal Disease 
A powerful, cost-effective new method for studying single-nucleotide polymorphisms (SNPs) is described. This method is based on the use of hairpin-shaped primers (HP), which give a sensitive and specific PCR amplification of each specific allele, without the use of costly fluorophore-labeled probes and any post-PCR manipulation. The amplification is monitored in real-time using SYBR Green I dye and takes only 2 h to yield results. The HP assay has a simple design and utilizes a conventional real-time PCR apparatus. The −44 C→G transversion in the DEFB1 gene (which encodes human β-defensin 1) has been previously associated with Candida carriage in oral epithelia. In this study, we analyzed the association between early-onset periodontal disease (EOP) and the −44 SNP. We used an HP assay to study the distribution of the −44 SNP in 264 human DNAs obtained from two cohorts of EOP patients and healthy controls from different ethnic backgrounds. The results indicate that the −44 SNP has a similar distribution between EOP and healthy patients, suggesting that it is not associated with the disease.
PMCID: PMC440632  PMID: 15242954
15.  Functional human T-cell immunity and osteoprotegerin ligand control alveolar bone destruction in periodontal infection  
Periodontitis, a prime cause of tooth loss in humans, is implicated in the increased risk of systemic diseases such as heart failure, stroke, and bacterial pneumonia. The mechanisms by which periodontitis and antibacterial immunity lead to alveolar bone and tooth loss are poorly understood. To study the human immune response to specific periodontal infections, we transplanted human peripheral blood lymphocytes (HuPBLs) from periodontitis patients into NOD/SCID mice. Oral challenge of HuPBL-NOD/SCID mice with Actinobacillus actinomycetemcomitans, a well-known Gram-negative anaerobic microorganism that causes human periodontitis, activates human CD4+ T cells in the periodontium and triggers local alveolar bone destruction. Human CD4+ T cells, but not CD8+ T cells or B cells, are identified as essential mediators of alveolar bone destruction. Stimulation of CD4+ T cells by A. actinomycetemcomitans induces production of osteoprotegerin ligand (OPG-L), a key modulator of osteoclastogenesis and osteoclast activation. In vivo inhibition of OPG-L function with the decoy receptor OPG diminishes alveolar bone destruction and reduces the number of periodontal osteoclasts after microbial challenge. These data imply that the molecular explanation for alveolar bone destruction observed in periodontal infections is mediated by microorganism-triggered induction of OPG-L expression on CD4+ T cells and the consequent activation of osteoclasts. Inhibition of OPG-L may thus have therapeutic value to prevent alveolar bone and/or tooth loss in human periodontitis.
This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, J. Clin. Invest. 106:R59–R67 (2000).
PMCID: PMC3102542  PMID: 10995794
16.  IFN-γ production in response to in vitro stimulation with collagen type II in rheumatoid arthritis is associated with HLA-DRB1*0401 and HLA-DQ8 
Arthritis Research  1999;2(1):75-84.
IFN-γ was measured in supernatants after in vitro stimulation of peripheral blood mononuclear cells with collagen type II (CII), purified protein derivative or influenza virus. IFN-γ production in response to CII was similar in rheumatoid arthritis (RA) patients and healthy control individuals. The IFN-γ response to purified protein derivative and influenza virus was lower in RA patients, reflecting a general T-cell hyporesponsiveness in RA. After recalculating the response to CII taking this hyporesponsiveness into account the CII response was higher in RA patients, and was associated with human leucocyte antigen (HLA)-DRB1*0401 and HLA-DQA1*0301-DQB1*0302 (HLA-DQ8). Rheumatoid arthritis patients with elevated serum levels of immunoglobulin (Ig)G anti-CII antibodies had lower CII-induced IFN-γ production than patients with low anti-CII levels. The relative increase in CII-reactivity in RA patients as compared with healthy control individuals, and the association of a higher response with RA-associated HLA haplotypes, suggest the existence of a potentially pathogenic cellular reactivity against CII in RA.
Despite much work over past decades, whether antigen-specific immune reactions occur in rheumatoid arthritis (RA) and to what extent such reactions are directed towards joint-specific autoantigens is still questionable. One strong indicator for antigenic involvement in RA is the fact that certain major histocompatibility complex (MHC) class II genotypes [human leucocyte antigen (HLA)-DR4 and HLA-DR1] predispose for the development of the disease [1]. In the present report, collagen type II (CII) was studied as a putative autoantigen on the basis of both clinical and experimental data that show an increased frequency of antibodies to CII in RA patients [2,3,4] and that show that CII can induce experimental arthritis [5].
It is evident from the literature that RA peripheral blood mononuclear cells (PBMCs) respond poorly to antigenic stimulation [6,7,8], and in particular evidence for a partial tolerization to CII has been presented [9]. The strategy of the present work has accordingly been to reinvestigate T-cell reactivity to CII in RA patients, to relate it to the response to commonly used recall antigens and to analyze IFN-γ responses as an alternative to proliferative responses.
To study cellular immune reactivity to CII in patients with RA and in healthy control individuals and to correlate this reactivity to HLA class II genotypes and to the presence of antibodies to CII in serum.
Forty-five patients who met the 1987 American College of Rheumatology classification criteria for RA [10] and 25 healthy control individuals of similar age and sex were included. Twenty-six of these patients who had low levels of anti-CII in serum were randomly chosen, whereas 19 patients with high anti-CII levels were identified by enzyme-linked immunosorbent assay (ELISA)-screening of 400 RA sera.
Heparinized blood was density gradient separated and PBMCs were cultured at 1 × 106/ml in RPMI-10% fetal calf serum with or without antigenic stimulation: native or denatured CII (100 μ g/ml), killed influenza virus (Vaxigrip, Pasteur Mérieux, Lyon, France; diluted 1 : 1000) or purified protein derivative (PPD; 10 μ g/ml). CII was heat-denatured in 56°C for 30 min.
Cell supernatants were collected after 7days and IFN-γ contents were analyzed using ELISA. HLA-DR and HLA-DQ genotyping was performed utilizing a polymerase chain reaction-based technique with sequence-specific oligonucleotide probe hybridization. Nonparametric statistical analyses were utilized throughout the study.
PBMCs from both RA patients and healthy control individuals responded with inteferon-γ production to the same degree to stimulation with native and denatured CII (Fig. 1a), giving median stimulation indexes with native CII of 4.6 for RA patients and 5.4 for healthy control individuals, and with denatured CII of 2.9 for RA patients and 2.6 for healthy control individuals. RA patients with elevated levels of anti-CII had a weaker IFN-γ response to both native and denatured CII than did healthy control individuals (P = 0.02 and 0.04, respectively).
Stimulation with the standard recall antigens PPD and killed influenza virus yielded a median stimulation index with PPD of 10.0 for RA patients and 51.3 for healthy control individuals and with influenza of 12.3 for RA patients and 25.7 for healthy, control individuals. The RA patients displayed markedly lower responsiveness to both PPD and killed influenza virus than did healthy control individuals (Fig. 1b). IFN-γ responses to all antigens were abrogated when coincubating with antibodies blocking MHC class II.
The low response to PPD and killed influenza virus in RA patients relative to that of healthy control individuals reflects a general downregulation of antigen-induced responsiveness of T cells from RA patients [6,7,8]. That no difference between the RA group and the control group was recorded in CII-induced IFN-γ production therefore indicates that there may be an underlying increased responsiveness to CII in RA patients, which is obscured by the general downregulation of T-cell responsiveness in these patients. In order to address this possibility, we calculated the fraction between individual values for the CII-induced IFN-γ production and the PPD-induced and killed influenza virus-induced IFN-γ production, and compared these fractions. A highly significant difference between the RA and healthy control groups was apparent after stimulation with both native CII and denatured CII when expressing the response as a fraction of that with PPD (Fig. 2a). Similar data were obtained using killed influenza virus-stimulated IFN-γ values as the denominator (Fig. 2b).
When comparing the compensated IFN-γ response to denatured CII stimulation between RA patients with different HLA genotypes, highly significant differences were evident, with HLA-DRB1*0401 patients having greater CII responsiveness than patients who lacked this genotype (Fig. 3a). HLA-DQ8 positive patients also displayed a high responsiveness to CII as compared with HLA-DQ8 negative RA patients (Fig. 3b). These associations between the relative T-cell reactivity to denatured CII and HLA class II genotypes were not seen in healthy control individuals. Similar results were achieved using influenza as denominator (P = 0.02 for HLA-DRB1*0401 and P = 0.01 for HLA-DQ8).
No reports have previously systematically taken the general T-cell hyporesponsiveness in RA into account when investigating specific T-cell responses in this disease. In order to address this issue we used the T-cell responses to PPD and killed influenza virus as reference antigens. This was made on the assumption that exposure to these antigens is similar in age-matched and sex-matched groups of RA patients and healthy control individuals. The concept of a general hyporesponsiveness in RA T cells has been documented in several previous reports, in which both nominal antigens [6,7,8] and mitogens [11,12,13] have been used. The fact that a similar functional downregulation in RA PBMCs was obtained with both PPD and killed influenza virus as reference antigens strengthens the validity of our approach.
We identified an association between the IFN-γ response to CII and HLA-DRB1*0401 and HLA-DQ8 in the RA patient group, which is of obvious interest because both these MHC class II alleles have been associated with high responsiveness to CII in transgenic mice that express these human MHC class II molecules [14,15]. There was no association between high anti-CII levels and shared epitope (HLA-DRB1*0401 or HLA-DRB1*0404).
CII, a major autoantigen candidate in RA, can elicit an IFN-γ response in vitro that is associated with HLA-DRB1*0401 and HLA-DQ8 in RA patients. This study, with a partly new methodological approach to a classical problem in RA, has provided some additional support to the notion that CII may be a target autoantigen of importance for a substantial group of RA patients. Continued efforts to identify mechanisms behind the general hyporesponsiveness to antigens in RA, as well as the mechanisms behind the potential partial anergy to CII, may provide us with better opportunities to study the specificity and pathophysiological relevance of anti-CII reactivity in RA.
PMCID: PMC17806  PMID: 11219392
collagen type II; human leucocyte antigen-DR; IFN-γ; rheumatoid arthritis; T cell
17.  Interleukin-5 is the predominant cytokine produced by peripheral blood mononuclear cells in alveolar echinococcosis. 
Infection and Immunity  1995;63(5):1688-1697.
An involvement of cellular immunity in alveolar echinococcosis is strongly suggested by the intense granulomatous infiltrations observed around the hepatic parasite lesions. However, the basis of cellular immunoregulation in patient with alveolar echinococcosis is poorly understood. The present report shows a comparative analysis of lymphoid cell function in peripheral blood mononuclear cells (PBMC) of 16 patients with alveolar echinococcosis and of healthy individuals. Our in vitro restimulation studies with crude Echinococcus multilocularis antigen demonstrated that PBMC from patients with alveolar echinococcosis were responsive to challenge with parasitic antigen as measured by lymphoid cell proliferation. In this system, we also evaluated cytokine expression at the gene and protein levels after stimulation with E. multilocularis antigen. Analysis of cytokine mRNA expression revealed distinct patterns of cytokine expression in patients and normal donors. By using reverse transcriptase PCR, we could demonstrate that the TH1 cytokine transcripts interleukin-2 (IL-2) and gamma interferon (IFN-gamma) are present in PBMC from patients with alveolar echinococcosis. Moreover, it was found that stimulation with E. multilocularis antigen induced or enhanced the expression of the TH2 cytokine IL-3, IL-4, IL-10, and especially IL-5 mRNAs in PBMC from 13 of 16 patients with alveolar echinococcosis. Two patients who were examined after radical surgery, as well as another patient with a stable course of the disease under continuous chemotherapy, were not able to generate the same pattern of cytokine response and had no evidence of IL-5 mRNA synthesis. In contrast to the frequent expression of TH2 cytokine mRNAs observed in patients with alveolar echinococcosis, PBMC cultures from normal donors showed prominent IL-2 and IFN-gamma mRNA expression but weak IL-3, IL-4, and IL-10 mRNA expression. Most interestingly, IL-5 mRNA was substantially absent in PBMC from healthy individuals. In accordance with the mRNA studies, it was found that E. multilocularis antigen induced the secretion of large amounts of IL-5 and intermediate amounts of IFN-gamma in patients with alveolar echinococcosis, whereas large amounts of IFN-gamma and no or threshold amounts of IL-5 were detected in supernatants from healthy individuals. Collectively, the present study provides the first evidence that a TH2 immune response is gradually activated during the course of E. multilocularis infection, indicating a critical role for IL-5 in the manifestation of human alveolar echinococcosis.
PMCID: PMC173211  PMID: 7729873
18.  Different Profile of Serum Leptin between Early Onset and Late Onset Preeclampsia 
Disease Markers  2014;2014:628476.
Aim. This study was designed to clarify the role of leptin and adiponectin in preeclampsia (PE) pathogenesis and different subtypes of preeclampsia. Method. This case control study was performed in 45 PE patients and 45 healthy controls matched for age, BMI, and ethnicity. Serum leptin and adiponectin levels were determined by enzyme linked immunosorbent assay (ELISA). Results. Maternal serum leptin and adiponectin were significantly higher in PE women than controls. Serum leptin was elevated in early onset preeclampsia (EOPE) and late onset preeclampsia (LOPE) compared to controls. Among PE patients, serum leptin was higher in EOPE than LOPE women. However, serum adiponectin was not different between EOPE and LOPE women. The serum leptin was significantly higher in severe PE than mild PE. The serum adiponectin was significantly elevated in severe PE compared to controls. Significant positive correlation was observed between leptin and adiponectin and also between leptin and BMI in controls. Moreover significant positive correlation was observed between adiponectin and BMI in PE patients and controls. Conclusion. The present study showed that serum leptin level may play a significant role as a biomarker to differentiate early and late onset PE and also its relation to BMI and severity of disease.
PMCID: PMC3925616  PMID: 24591763
19.  Specificity of T cells in synovial fluid: high frequencies of CD8+ T cells that are specific for certain viral epitopes 
Arthritis Research  2000;2(2):154-164.
CD8+ T cells dominate the lymphocyte population in synovial fluid in chronic inflammatory arthritis. It is known that these CD8+ T cells are often clonally or oligoclonally expanded, but their specificity and their relevance to the pathogenesis of joint disease has remained unclear. We found that as many as 15.5% of synovial CD8+ T cells may be specific for a single epitope from an Epstein-Barr virus lytic cycle protein. The virus-specific T cells within the joint showed increased expression of markers of activation and differentiation compared with those in the periphery, and retained their functional capacity to secrete proinflammatory cytokines on stimulation. These activated, virus-specific CD8+ T cells could therefore interact with synoviocytes, either by cell-cell contact or by a cytokine network, and play a 'bystander' role in the maintenance of inflammation in patients with arthritis.
Epstein-Barr virus (EBV) is transmitted orally, replicates in the oropharynx and establishes life-long latency in human B lymphocytes. T-cell responses to latent and lytic/replicative cycle proteins are readily detectable in peripheral blood from healthy EBV-seropositive individuals. EBV has also been detected within synovial tissue, and T-cell responses to EBV lytic proteins have been reported in synovial fluid from a patient with rheumatoid arthritis (RA). This raises the question regarding whether T cells specific for certain viruses might be present at high frequencies within synovial fluid and whether such T cells might be activated or able to secrete cytokines. If so, they might play a 'bystander' role in the pathogenesis of inflammatory joint disease.
To quantify and characterize T cells that are specific for epitopes from EBV, cytomegalovirus (CMV) and influenza in peripheral blood and synovial fluid from patients with arthritis.
Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were obtained from patients with inflammatory arthritis (including those with RA, osteoarthritis, psoriatic arthritis and reactive arthritis). Samples from human leucocyte antigen (HLA)-A2-positive donors were stained with fluorescent-labelled tetramers of HLA-A2 complexed with the GLCTLVAML peptide epitope from the EBV lytic cycle protein BMLF1, the GILGFVFTL peptide epitope from the influenza A matrix protein, or the NLVPMVATV epitope from the CMV pp65 protein. Samples from HLA-B8-positive donors were stained with fluorescent-labelled tetramers of HLA-B8 complexed with the RAKFKQLL peptide epitope from the EBV lytic protein BZLF1 or the FLRGRAYGL peptide epitope from the EBV latent protein EBNA3A. All samples were costained with an antibody specific for CD8. CD4+ T cells were not analyzed. Selected samples were costained with antibodies specific for cell-surface glycoproteins, in order to determine the phenotype of the T cells within the joint and the periphery. Functional assays to detect release of IFN-γ or tumour necrosis factor (TNF)-α were also performed on some samples.
The first group of 15 patients included 10 patients with RA, one patient with reactive arthritis, one patient with psoriatic arthritis and three patients with osteoarthritis. Of these, 11 were HLA-A2 positive and five were HLA-B8 positive. We used HLA-peptide tetrameric complexes to analyze the frequency of EBV-specific T cells in PBMCs and SFMCs (Figs 1 and 2). Clear enrichment of CD8+ T cells specific for epitopes from the EBV lytic cycle proteins was seen within synovial fluid from almost all donors studied, including patients with psoriatic arthritis and osteoarthritis and those with RA. In donor RhA6, 9.5% of CD8+ SFMCs were specific for the HLA-A2 restricted GLCTLVAML epitope, compared with 0.5% of CD8+ PBMCs. Likewise in a donor with osteoarthritis (NR4), 15.5% of CD8+ SFMCs were specific for the HLA-B8-restricted RAKFKQLL epitope, compared with 0.4% of CD8+ PBMCs. In contrast, we did not find enrichment of T cells specific for the HLA-B8-restricted FLRGRAYGL epitope (from the latent protein EBNA3A) within SFMCs compared with PBMCs in any donors. In selected individuals we performed ELISpot assays to detect IFN-γ secreted by SFMCs and PBMCs after a short incubation in vitro with peptide epitopes from EBV lytic proteins. These assays confirmed enrichment of T cells specific for epitopes from EBV lytic proteins within synovial fluid and showed that subpopulations of these cells were able to secrete proinflammatory cytokines after short-term stimulation.
We used a HLA-A2/GILGFVFTL tetramer to stain PBMCs and SFMCs from six HLA-A2-positive patients. The proportion of T cells specific for this influenza epitope was low (<0.2%) in all donors studied, and we did not find any enrichment within SFMCs.
We had access to SFMCs only from a second group of four HLA-A2-positive patients with RA. A tetramer of HLA-A2 complexed to the NLVPMVATV epitope from the CMV pp65 protein reacted with subpopulations of CD8+ SFMCs in all four donors, with frequencies of 0.2, 0.5, 2.3 and 13.9%. SFMCs from all four donors secreted TNF after short-term incubation with COS cells transfected with HLA-A2 and pp65 complementary DNA. We analyzed the phenotype of virus-specific cells within PBMCs and SFMCs in three donors. The SFMC virus-specific T cells were more highly activated than those in PBMCs, as evidenced by expression of high levels of CD69 and HLA-DR. A greater proportion of SFMCs were CD38+, CD62L low, CD45RO bright, CD45RA dim, CD57+ and CD28- when compared with PBMCs.
This work shows that T cells specific for certain epitopes from viral proteins are present at very high frequencies (up to 15.5% of CD8+ T cells) within SFMCs taken from patients with inflammatory joint disease. This enrichment does not reflect a generalized enrichment for the 'memory pool' of T cells; we did not find enrichment of T cells specific for the GILGFVFTL epitope from influenza A or for the FLRGRAYGL epitope from the EBV latent protein EBNA3A, whereas we found clear enrichment of T cells specific for the GLCTLVAML epitope from the EBV lytic protein BMLF1 and for the RAKFKQLL epitope from the EBV lytic protein BZLF1.
The enrichment might reflect preferential recruitment of subpopulations of virus-specific T cells, perhaps based on expression of selectins, chemokine receptors or integrins. Alternatively, T cells specific for certain viral epitopes may be stimulated to proliferate within the joint, by viral antigens themselves or by cross-reactive self-antigens. Finally, it is theoretically possible that subpopulations of T cells within the joint are preferentially protected from apoptotic cell death. Whatever the explanation, the virus-specific T cells are present at high frequency, are activated and are able to secrete proinflammatory cytokines. They could potentially interact with synoviocytes and contribute to the maintenance of inflammation within joints in many different forms of inflammatory arthritis.
PMCID: PMC17809  PMID: 11062606
CD8+ T cell; Epstein-Barr virus lytic cycle; human leucocyte antigen peptide tetrameric complex; rheumatoid arthritis; viral immunity
20.  Antibody and T Cell Responses to Fusobacterium nucleatum and Treponema denticola in Health and Chronic Periodontitis 
PLoS ONE  2013;8(1):e53703.
The characteristics of the T cell response to the members of oral flora are poorly understood. We characterized the antibody and T cell responses to FadA and Td92, adhesins from Fusobacterium nucleatum, an oral commensal, and Treponema denticola, a periodontal pathogen, respectively. Peripheral blood and saliva were obtained from healthy individuals and patients with untreated chronic periodontitis (CP, n = 11 paris) and after successful treatment of the disease (n = 9). The levels of antigen-specific antibody were measured by ELISA. In plasma, IgG1 was the most abundant isotype of Ab for both Ags, followed by IgA and then IgG4. The levels of FadA-specific salivary IgA (sIgA) were higher than Td92-specific sIgA and the FadA-specific IgA levels observed in plasma. However, the periodontal health status of the individuals did not affect the levels of FadA- or Td92-specific antibody. Even healthy individuals contained FadA- and Td92-specific CD4+ T cells, as determined by the detection of intracytoplasmic CD154 after short-term in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with the antigens. Patients with CP tended to possess increased numbers of FadA- and Td92-specific CD4+ T cells but reduced numbers of Td92-specific Foxp3+CD4+ Tregs than the healthy subjects. Both FadA and Td92 induced the production of IFNγ and IL-10 but inhibited the secretion of IL-4 by PBMCs. In conclusion, F. nucleatum induced Th3 (sIgA)- and Th1 (IFNγ and IgG1)-dominant immune responses, whereas T. denticola induced a Th1 (IFNγ and IgG1)-dominant response. This IFNγ-dominant cytokine response was impaired in CP patients, and the Td92-induced IFNγ levels were negatively associated with periodontal destruction in patients. These findings may provide new insights into the homeostatic interaction between the immune system and oral bacteria and the pathogenesis of periodontitis.
PMCID: PMC3546045  PMID: 23335969
21.  Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects 
Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.
PMCID: PMC3854147  PMID: 22850872
Antimicrobial peptides; Nitric oxide; Neutrophils; Periodontitis; Innate immunity
22.  Recombinant Actinobacillus actinomycetemcomitans Cytolethal Distending Toxin Proteins Are Required To Interact To Inhibit Human Cell Cycle Progression and To Stimulate Human Leukocyte Cytokine Synthesis 
Infection and Immunity  2001;69(9):5925-5930.
It has recently been discovered that Actinobacillus actinomycetemcomitans, an oral bacterium causing periodontitis, produces cytolethal distending toxin (CDT), a cell cycle-modulating toxin that has three protein subunits: CdtA, CdtB, and CdtC. In this study, we have cloned and expressed each toxin gene from A. actinomycetemcomitans in Escherichia coli and purified the recombinant Cdt proteins to homogeneity. Individual Cdt proteins failed to induce cell cycle arrest of the human epithelial cell line HEp-2. The only combinations of toxin proteins causing cell cycle arrest were the presence of all three Cdt proteins and the combination of CdtB and CdtC. A similar experimental protocol was used to determine if recombinant Cdt proteins were able to induce human peripheral blood mononuclear cells (PBMCs) to produce cytokines. The individual Cdt proteins were able to induce the synthesis by PBMCs of interleukin-1β (IL-1β), IL-6, and IL-8 but not of tumor necrosis factor alpha, IL-12, or granulocyte-macrophage colony-stimulating factor, with CdtC being the most potent and CdtB being the least potent cytokine inducer. There was evidence of synergism between these Cdt proteins in the stimulation of cytokine production, most markedly with gamma interferon, which required the minimum interaction of CdtB and -C to stimulate production.
PMCID: PMC98715  PMID: 11500475
23.  Cytokine interactions in human immunodeficiency virus-infected individuals: roles of interleukin (IL)-2, IL-12, and IL-15 
The Journal of Experimental Medicine  1995;182(4):1067-1077.
Cytokines have been shown to be powerful regulators of the immune response. In this study, we analyze the effect that the newly recognized cytokine interleukin (IL)-15 has on proliferation and cytokine induction using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from patients infected with human immunodeficiency virus (HIV) who are at various stages in their disease. We observed that IL-15 enhances the proliferative response in a dose-dependent manner from PBMCs of HIV-infected individuals when stimulated by polyclonal mitogen, tetanus toxoid, or HIV-specific antigen. The effects of exogenous IL-15 are substantially diminished by adding a neutralizing antibody to the beta chain of the IL-2 receptor. Moreover, the ability of IL-15 to increase proliferation is enhanced by the presence of endogenous IL-2 produced in the cultures. The effect that exogenous IL-15 had on IL-2, IL-4, and interferon (IFN)-gamma induction from PBMC's or CD4+ T cells in response to mitogen or tetanus toxoid was also examined. This was compared to the effect that exogenous IL-2 and IL-12 had under the same conditions. Addition of IL- 2 or IL-15 to short-term in vitro cultures of either PBMCs or CD4+ T cells had little effect on IL-2, IL-4, or IFN-gamma production. By contrast, IL-12 caused substantial enhancement of both IL-2 and IFN- gamma production from these cultures. The role that endogenous cytokines have on IFN-gamma induction was also studied. Addition of a neutralizing antibody to the alpha chain of the IL-2 receptor or IL-12 to antigen stimulated cultures caused a striking decrease in IFN-gamma production. Neutralization of endogenous IL-15 also resulted in diminished IFN-gamma production from cultures stimulated with mitogen. IL-4 and IFN-gamma protein production by PBMCs and CD4+ T cells stimulated with mitogen was assessed to see if we could detect a specific bias of cytokine production. Small amounts of IL-4 were detected from CD4+ T cells but not PBMCs from most individuals tested. IFN-gamma and IL-2, however, were also produced from these same cultures. These results further elucidate the mechanism of cytokine regulation in HIV-infected individuals, and they provide evidence that IL-15 may be a useful immune modulator.
PMCID: PMC2192305  PMID: 7561680
24.  Differential Production of Systemic and Intralesional Gamma Interferon and Interleukin-10 in Nodular and Ulcerative Forms of Buruli Disease  
Infection and Immunity  2004;72(2):958-965.
Buruli disease, caused by Mycobacterium ulcerans, is the third most important mycobacterial disease in humans besides tuberculosis and leprosy. We have compared systemic and intralesional cytokine production in patients presenting with a nodular form and a necrotizing, ulcerative form of the disease. Gamma interferon (IFN-γ) levels in response to whole M. ulcerans and Mycobacterium bovis BCG bacilli and in response to purified Ag85 protein from BCG were lower in peripheral blood mononuclear cells (PBMC) cultures from Buruli disease patients than in PBMC from healthy purified protein derivative-positive contacts. Interleukin-4 (IL-4) and IL-13 content was below the detection threshold in these PBMC cultures. IFN-γ production after stimulation with M. ulcerans was significantly lower (P < 0.05) in PBMC cultures from patients with ulcers than in those from patients with nodules. On the other hand, PBMC from Buruli disease patients produced significant levels of IL-10 in response to M. ulcerans (but not to M. bovis BCG) and production was highest in patients with the ulcerative form. Third, semiquantitative reverse transcription-PCR analysis demonstrated a similar difference in the local, intralesional cytokine profile for the two forms of the disease: high IFN-γ but low IL-10 mRNA levels in nodular lesions and high IL-10 but low IFN-γ mRNA levels in ulcerative lesions. Intralesional IL-4 and IL-13 mRNA levels were low and only detected in patients with the ulcerative form. Our results indicate, although they do not formally prove, that production of IL-10 rather than production of IL-4 or IL-13 by Th2-type T cells may be involved in the low M. ulcerans-specific IFN-γ response in Buruli disease patients.
PMCID: PMC321599  PMID: 14742541
25.  The importance of the presence of aggregatibacter actinomycetemcomitans in sulcus gingivalis of patients with cardiovascular diseases 
Over-replication of periodontal pathogens in the periodontium induces production of proinflammatory cytokines and C-reactive protein that can stimulate systemic inflammatory status and can initiate atherosclerosis and its consequences. In our pilot study we examined whether periodontal status and serum levels of interleukin-6 and C-reactive protein are associated with the presence of Aggregatibacter actinomycetemcomitans in the periodontium of patients with cardiovascular diseases (CVD).
We randomly selected 38 of 166 outpatients with CVD, of which 21 patients had chronic ischemic heart disease (IHD) only and 17 had both IHD and essential hypertension (HT). The presence of Aggregatibacter actinomycetemcomitans (A.a.) in the periodontium evaluated by PCR was compared with the values of periodontal indices, namely probe depth (PD) and Community Periodontal Index of Treatment Need (CPITN), as well as with interleukin-6 (IL-6) and CRP serum levels.
When comparing A.a.-positive and A.a.-negative groups of patients, no statistically significant differences were noticed as to the age and values of PD and CPITN, respectively. However, the proportion of CRP and IL-6 positive values was significantly higher (p≤0.001) among A.a.-positive than in A.a.-negative patients.
The presence of A.a. in patients with CVD may be associated with significantly higher serum levels of some proinflammatory markers.
PMCID: PMC3539491  PMID: 22037744
cytokines; CRP; periodontal indexes; periodontal diseases

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