Impact of RYGB surgery on plasma immunoglobulins: association between blood pressure and glucose levels six months after surgery.

We aimed to study levels of natural antibodies in plasma, and their associations to clinical and fecal biomarkers, before and 6 months after Roux-en-Y gastric bypass (RYGB) surgery. Thirty individuals with obesity [16 type 2 diabetic, 14 non-diabetic (ND)] had RYGB surgery. Total plasma IgA, IgG and IgM antibody levels and specific antibodies to oxidized low-density lipoprotein (oxLDL), malondialdehyde-acetaldehyde adducts, Porphyromonas gingivalis gingipain A hemagglutinin domain (Rgp44), and phosphocholine were measured using chemiluminescence immunoassay. Associations between plasma and fecal antibodies as well as clinical markers were analyzed. RYGB surgery reduced blood pressure, and the glycemic state was improved. A higher level of diastolic blood pressure was associated with lower plasma antibodies to oxLDL after surgery. Also, lower level of glucose markers associated with lower level of plasma antibodies to bacterial virulence factors. Antibodies to oxLDL decreased after surgery, and positive association between active serum lipopolysaccharide and specific oxLDL antibodies was detected. Total IgG levels decreased after surgery, but only in ND individuals. Reduced level of total plasma IgG, improved state of hypertension and hyperglycemia and their associations with decreased levels of specific antibodies in plasma, suggest an improved state of systemic inflammation after RYGB surgery.

Dissociation of murine oral mucosal tissues for single cell applications.

Single-cell RNA sequencing and flow cytometry approaches have been instrumental in understanding cellular states within various tissues and organs. However, tissue dissociation methods can potentially alter results and create bias due to preferential recovery of particular cell types. Here we present efforts to optimize methods for dissociation of murine oral mucosal tissues and provide three different protocols that can be utilized to isolate major cell populations in the oral mucosa. These methods can be used both in health and in states of inflammation, such as periodontitis. The optimized protocols use different enzymatic approaches (collagenase II, collagenase IV and the Miltenyi whole skin dissociation kit) and yield preferential recovery of immune, stromal and epithelial cells, respectively. We suggest choosing the dissociation method based on the cell population of interest to study, while understanding the limitations of each approach.

Total fecal IgA levels increase and natural IgM antibodies decrease after gastric bypass surgery.

Obesity is associated with low-grade inflammation and increased systemic oxidative stress. Roux-en-Y gastric bypass (RYGB) surgery is known to ameliorate the obesity-induced metabolic dysfunctions. We aimed to study the levels of natural antibodies in feces, before and 6 months after RYGB surgery in obese individuals with and without type 2 diabetes (T2D). Sixteen individuals with T2D and 14 non-diabetic (ND) individuals were operated. Total IgA, IgG and IgM antibody levels and specific antibodies to oxidized low-density lipoprotein (oxLDL), malondialdehyde-acetaldehyde adducts (MAA adducts), Porphyromonas gingivalis gingipain A hemagglutinin domain (Rgp44) and phosphocholine (PCho) were measured using chemiluminescence immunoassay. Total fecal IgA was elevated, while total IgM and IgG were not affected by the surgery. Fecal natural IgM specific to oxLDL decreased significantly in both T2D and ND individuals, while fecal IgM to Rgp44 and PCho decreased significantly in T2D individuals. A decrease in IgG to MAA-LDL, Rgp44 and PCho was detected. RYGB surgery increases the levels of total fecal IgA and decreases fecal natural IgG and IgM antibodies specific to oxLDL. Natural antibodies and IgA are important in maintaining the normal gut homeostasis and first-line defense against microbes, and their production is markedly altered with RYGB surgery.

Salivary IgA antibody to malondialdehyde-acetaldehyde associates with mild periodontal pocket depth.

OBJECTIVE: Oxidized epitopes such as malondialdehyde-acetaldehyde (MAA) play a crucial role in the progression of atherosclerosis through activation of the humoral immune response. The exact mechanism of the association between atherosclerosis and periodontal diseases is not fully understood. The aim of the current study is to evaluate the association of oral humoral immune response to oxidized epitopes with parameters of periodontal disease. MATERIALS AND METHODS: The Parogene cohort consist of patients who have undergone coronary angiography due to cardiac symptoms. In this study, 423 patients were randomly selected for an extensive oral examination. Salivary Immunoglobulin A to oxidized epitopes and bacterial antigens was determined by chemiluminescence immunoassay. RESULTS: In a binary logistic regression model adjusted with periodontal disease confounders, periodontal pocket depth (PPD) 4-5 mm associated with salivary IgA antibodies to MAA-LDL (p = 0.034), heat shock protein 60 of Aggregatibacter actinomycetemcomitans (p = 0.045), Porphyromonas gingivalis (p = 0.045), A. actinomycetemcomitans (p = 0.005), P. intermedia (p = 0.020), and total IgA (p = 0.003). CONCLUSIONS: The current study shows the association of salivary IgA to MAA-LDL with PPD 4-5 mm in a cohort of patients with chronic coronary artery disease. Humoral immune cross-reactivation to oxidized epitopes such MAA-LDL could partly explain the link of periodontitis with systemic diseases.

Compensatory IgM to the Rescue: Patients with Selective IgA Deficiency Have Increased Natural IgM Antibodies to MAA-LDL and No Changes in Oral Microbiota.

IgA is the most abundant Ab in the human body. However, most patients with selective IgA deficiency (SIgAD) are asymptomatic. IgM, and to lesser extent IgG Abs, are generally presumed to compensate for the lack of IgA in SIgAD by multiplying and adopting functions of IgA. We used data from the Northern Finland Birth Cohort 1966 to investigate whether SIgAD patients have differences in levels of natural Abs to oxidized epitopes compared with 20 randomly selected healthy controls. First, we screened the saliva and serum samples from the Northern Finland Birth Cohort 1966 cohort (n = 1610) for IgA concentration. We detected five IgA-deficient subjects, yielding a prevalence of 0.3%, which is consistent with the general prevalence of 0.25% in the Finnish population. To detect natural Abs, we used malondialdehyde acetaldehyde-low-density lipoprotein (MAA-LDL), an Ag known to bind natural Abs. In this study, we show that natural secretory IgM and IgG Abs to MAA-DL were significantly increased in subjects with SIgAD. Given that secretory IgA is an important part of mucosal immune defense and that, in the gut microbiota, dysbiosis with SIgAD patients has been observed, we characterized the oral bacterial microbiota of the subjects with and without SIgAD using high-throughput 16S rRNA gene sequencing. We found no significant alterations in diversity and composition of the oral microbiota in subjects with SIgAD. Our data suggest that increased levels of secretory natural Abs in patients with SIgAD could be a compensatory mechanism, providing alternative first-line defense against infections and adjusting mucosal milieu to maintain a healthy oral microbiota.

Existence of natural mouse IgG mAbs recognising epitopes shared by malondialdehyde acetaldehyde adducts and Porphyromonas gingivalis.

Natural Abs are produced by B lymphocytes in the absence of external Ag stimulation. They recognise self, altered self and foreign Ags, comprising an important first-line defence against invading pathogens and serving as innate recognition receptors for tissue homeostasis. Natural IgG Abs have been found in newborns and uninfected individuals. Yet, their physiological role remains unclear. Previously, no natural IgG Abs to oxidation-specific epitopes have been reported. Here, we show the cloning and characterisation of mouse IgG mAbs against malondialdehyde acetaldehyde (MAA)-modified low-density lipoprotein. Sequence analysis reveals high homology with germline genes, suggesting that they are natural. Further investigation shows that the MAA-specific natural IgG Abs cross-react with the major periodontal pathogen Porphyromonas gingivalis and recognise its principle virulence factors gingipain Kgp and long fimbriae. The study provides evidence that natural IgGs may play an important role in innate immune defence and in regulation of tissue homeostasis by recognising and removing invading pathogens and/or modified self-Ags, thus being involved in the development of periodontitis and atherosclerosis.

Immunological and Microbiological Profiling of Cumulative Risk Score for Periodontitis.

The cumulative risk score (CRS) is a mathematical salivary diagnostic model to define an individual's risk of having periodontitis. In order to further validate this salivary biomarker, we investigated how periodontal bacteria, lipopolysaccharide (LPS), and systemic and local host immune responses relate to CRS. Subgingival plaque, saliva, and serum samples collected from 445 individuals were used in the analyses. Plaque levels of 28 microbial species, especially those of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Porphyromonas endodontalis, Prevotella intermedia, and Tannerella forsythia, and serum and salivary levels of IgA and IgG against these five species were determined. Additionally, LPS activity was measured. High CRS associated strongly with all IgA/IgG antibody and LPS levels in saliva, whereas in serum the associations were not that obvious. In the final logistic regression model, the best predictors of high CRS were saliva IgA burden against the five species (OR 7.04, 95% CI 2.25-22.0), IgG burden (3.79, 1.78-8.08), LPS (2.19, 1.38-3.47), and the sum of 17 subgingival Gram-negative species (6.19, 2.10-18.3). CRS is strongly associated with microbial biomarker species of periodontitis and salivary humoral immune responses against them.

Humoral immune response to heat shock protein 60 of Aggregatibacter actinomycetemcomitans and cross-reactivity with malondialdehyde acetaldehyde-modified LDL.

Atherosclerosis is a chronic inflammatory disease and major cause of mortality worldwide. One of the crucial steps for atherosclerotic plaque development is oxidation of low-density lipoprotein (LDL). Through the oxidation, highly immunogenic epitopes are created and the immune system is activated. Association between atherosclerosis and periodontal diseases is well documented, and one of the main oral pathogens common in periodontitis is Aggregatibacter actinomycetemcomitans (Aa). Heat shock protein 60 (HSP60) is an important virulence factor for Aa bacteria and a strong activator of the immune system. Cross-reactivity of HSP60 and oxidized LDL (OxLDL) antibodies could be a potential mechanism in the progression of atherosclerosis and one possible link between atherosclerosis and periodontitis. Human plasma samples from neonates and mothers were analyzed to determine if antibody titer to Aa-HSP60 protein is already present in newborns. Further objectives were to characterize antibody response in Aa-HSP60 immunized mice and to determine possible antibody cross-reaction with oxidized LDL. We demonstrated that newborns already have IgM antibody levels to Aa-HSP60. We also showed that in mice, Aa-HSP60 immunization provoked IgG and IgM antibody response not only to Aa-HSP60 but also to malondialdehyde acetaldehyde-modified LDL (MAA-LDL). Competition assay revealed that the antibodies were specific to Aa-HSP60 and cross-reacted with MAA-LDL. Our results suggest a possibility of molecular mimicry between Aa-HSP60 and MAA-LDL, making it intriguing to speculate on the role of HSP60 protein in atherosclerosis that manifests at young age.

Saliva and Serum Immune Responses in Apical Periodontitis.

Apical periodontitis is an inflammatory reaction at the apex of an infected tooth. Its microbiota resembles that of marginal periodontitis and may induce local and systemic antibodies binding to bacteria- and host-derived epitopes. Our aim was to investigate the features of the adaptive immune response in apical periodontitis. The present Parogene cohort (n = 453) comprises patients with cardiac symptoms. Clinical and radiographic oral examination was performed to diagnose apical and marginal periodontitis. A three-category endodontic lesion score was designed. Antibodies binding to the bacteria- and host-derived epitopes were determined from saliva and serum, and bacterial compositions were examined from saliva and subgingival samples. The significant ORs (95% CI) for the highest endodontic scores were observed for saliva IgA and IgG to bacterial antigens (2.90 (1.01-8.33) and 4.91 (2.48-9.71)/log10 unit), saliva cross-reacting IgG (2.10 (1.48-2.97)), serum IgG to bacterial antigens (4.66 (1.22-10.1)), and Gram-negative subgingival species (1.98 (1.16-3.37)). In a subgroup without marginal periodontitis, only saliva IgG against bacterial antigens associated with untreated apical periodontitis (4.77 (1.05-21.7)). Apical periodontitis associates with versatile adaptive immune responses against both bacterial- and host-derived epitopes independently of marginal periodontitis. Saliva immunoglobulins could be useful biomarkers of oral infections including apical periodontitis-a putative risk factor for systemic diseases.

Potential role of nuclear magnetic resonance spectroscopy to identify salivary metabolite alterations in patients with head and neck cancer.

The analysis of the salivary metabolomic profile may offer an early phase approach to assess the changes associated with a wide range of diseases including head and neck cancer. The aim of the present study was to investigate the potential of nuclear magnetic resonance (NMR) spectroscopy for detecting the salivary metabolic changes associated with head and neck squamous cell carcinoma (HNSCC). Unstimulated whole-mouth saliva samples collected from HNSCC patients (primary tumour was located either in the larynx or in the oral cavity) and healthy controls were analysed by 1H-NMR spectroscopy. Reliably identified salivary metabolites were quantified and the determined concentration values were compared group-wise using a Mann-Whitney U-test. Multivariate discrimination function analysis (DFA) was conducted to identify such a combination of metabolites, when considered together, that gives maximum discrimination between the groups. HNSCC patients exhibited significantly increased concentrations of 1,2-propanediol (P=0.032) and fucose (P=0.003), while proline levels were significantly decreased (P=0.043). In the DFA model, the most powerful discrimination was achieved when fucose, glycine, methanol and proline were considered as combined biomarkers, resulting in a correct classification rate of 92.1%, sensitivity of 87.5% and specificity of 93.3%. To conclude, NMR spectrometric analysis was revealed to be a feasible approach to study the metabolome of saliva that is sensitive to metabolic changes in HNSCC and straightforward to collect in a non-invasive manner. Salivary fucose was of particular interest and therefore, controlled longitudinal studies are required to assess its clinical relevance as a diagnostic biomarker in HNSCC.

Immunization with gingipain A hemagglutinin domain of Porphyromonas gingivalis induces IgM antibodies binding to malondialdehyde-acetaldehyde modified low-density lipoprotein.

Treatment of periodontitis has beneficial effects on systemic inflammation markers that relate to progression of atherosclerosis. We aimed to investigate whether immunization with A hemagglutinin domain (Rgp44) of Porphyromonas gingivalis (Pg), a major etiologic agent of periodontitis, would lead to an antibody response cross-reacting with oxidized low-density lipoprotein (OxLDL) and how it would affect the progression of atherosclerosis in low-density lipoprotein receptor-deficient (LDLR-/-) mice. The data revealed a prominent IgM but not IgG response to malondialdehyde-acetaldehyde modified LDL (MAA-LDL) after Rgp44 and Pg immunizations, implying that Rgp44/Pg and MAA adducts may share cross-reactive epitopes that prompt IgM antibody production and consequently confer atheroprotection. A significant negative association was observed between atherosclerotic lesion and plasma IgA to Rgp44 in Rgp44 immunized mice, supporting further the anti-atherogenic effect of Rgp44 immunization. Plasma IgA levels to Rgp44 and to Pg in both Rgp44- and Pg-immunized mice were significantly higher than those in saline control, suggesting that IgA to Rgp44 could be a surrogate marker of immunization in Pg-immunized mice. Distinct antibody responses in plasma IgA levels to MAA-LDL, to Pg lipopolysaccharides (Pg-LPS), and to phosphocholine (PCho) were observed after Rgp44 and Pg immunizations, indicating that different immunogenic components between Rpg44 and Pg may behave differently in regard of their roles in the development of atherosclerosis. Immunization with Rgp44 also displayed atheroprotective features in modulation of plaque size through association with plasma levels of IL-1α whereas whole Pg bacteria achieved through regulation of anti-inflammatory cytokine levels of IL-5 and IL-10. The present study may contribute to refining therapeutic approaches aiming to modulate immune responses and inflammatory/anti-inflammatory processes in atherosclerosis.

IgA antibodies to phosphocholine associate with long-term cardiovascular disease risk.

BACKGROUND AND AIMS: Antibodies to phosphocholine and oxidized LDL (oxLDL) are proposed to modify progression of atherosclerosis. We investigated the prognostic value of antibodies to phosphocholine (PCho), Streptococcus pneumoniae cell wall polysaccharide (CWPS) and oxLDL in defining long-term CVD survival. METHODS: CVD incidence was followed for 18 years and analyzed with baseline plasma IgM, IgG and IgA antibody levels to PCho, CWPS and oxLDL in 1044 subjects of Oulu Project Elucidating Risk of Atherosclerosis study (OPERA). RESULTS: During the follow-up period, 195 subjects (18.7%) had a CVD event. Cox model with ACC/AHA CVD adjustments (ASCVD) showed that IgA levels to PCho and IgA to CWPS were statistically significant factors predicting CVD risk. IgM and IgG antibodies to PCho, CWPS and oxLDL had no effect on CVD risk after adjusting for other risk factors. Net reclassification improvement (categories: 17-year risk <15%, 15-30%, >30%), was 0.06 (-0.001-0.12, p < 0.054), and IDI was 0.0124 (0.0036-0.0211, p < 0.006) with IgA-PCho added to the ASCVD risk model. Seventeen (9.4%) study subjects with CVD events were correctly reclassified into higher risk category while 9 (5.0%) subjects were classified into lower risk category. Among the non-cases, 58 (8.7%) subjects were correctly reclassified into lower risk, and 46 (5.9%) were reclassified into higher risk category. CONCLUSIONS: Plasma IgA antibodies to PCho and Streptococcus pneumoniae CWPS are significant predictors of long-term CVD risk. Additional studies on the role of IgA antibodies in atherogenesis and CVD are warranted.

Cross-reactive saliva IgA antibodies to oxidized LDL and periodontal pathogens in humans.

AIM: Oxidized low-density lipoproteins (oxLDL) are formed as a result of lipid peroxidation and are highly immunogenic and proatherogenic. In this study, saliva antibodies binding to oxLDL, Porphyromonas gingivalis (Pg) and Aggregatibacter actinomycetemcomitans (Aa) were characterized and their cross-reactivity was evaluated. MATERIALS AND METHODS: Resting and stimulated saliva samples were collected from 36 healthy adults (mean age 26 years). Saliva IgA, IgG and IgM autoantibody levels to copper oxidized LDL (CuOx-LDL) and malondialdehyde acetaldehyde-modified LDL (MAA-LDL) were determined with chemiluminescence immunoassay. RESULTS: Saliva IgA and IgG antibodies binding to MAA-LDL and CuOx-LDL were detected in all samples and they were associated with the saliva levels of IgA and IgG to P. gingivalis and A. actinomycetemcomitans. Competitive immunoassay showed that saliva antibodies to MAA-LDL cross-reacted specifically with P. gingivalis. The autoantibody levels to oxLDL in saliva were not associated with the autoantibody levels to oxLDL in plasma or with saliva apolipoprotein B 100 levels. CONCLUSIONS: Saliva contains IgA and IgG binding to oxLDL, which showed cross-reactive properties with the periodontal pathogens Porphyromonas gingivalis (P.g). The data suggest that secretory IgA to P.g may participate in immune reactions involved in LDL oxidation through molecular mimicry.

Characterization of a natural mouse monoclonal antibody recognizing epitopes shared by oxidized low-density lipoprotein and chaperonin 60 of Aggregatibacter actinomycetemcomitans.

Natural antibodies are predominantly antibodies of the IgM isotype present in the circulation of all vertebrates that have not been previously exposed to exogenous antigens. They are often directed against highly conserved epitopes and bind to ligands of varying chemical composition with low affinity. In this study we cloned and characterized a natural mouse monoclonal IgM antibody selected by binding to malondialdehyde acetaldehyde epitopes on low-density lipoprotein (LDL). Interestingly, the IgM antibody cross-reacted with Aggregatibacter actinomycetemcomitans (Aa) bacteria, a key pathogenic microbe in periodontitis reported to be associated with risk factor for atherosclerosis, thus being named as Aa_Mab. It is more intriguing that the binding molecule of Aa to Aa_Mab IgM was found to be Aa chaperonin 60 or HSP60, a member of heat-shock protein family, behaving not only as a chaperone for correct protein folding but also as a powerful virulence factor of the bacteria for inducing bone resorption and as a putative pathogenic factor in atherosclerosis. The findings will highlight the question of whether molecular mimicry between pathogen components and oxidized LDL could lead to atheroprotective immune activity, and also would be of great importance in potential application of immune response-based preventive and therapeutic strategies against atherosclerosis and periodontal disease.