Free light chains as an emerging biomarker in saliva::biological variability and comparisons with salivary IgA and steroid hormones

BackgroundSalivary free light chains (FLCs) are an emerging biomarker in health and behavioural research. However, little is known regarding biological variability of salivary FLCs and how they relate to other established salivary biomarkers. This study aimed to investigate the diurnal and day-to-day variation of salivary FLCs and their relationship with salivary IgA and steroid hormones.MethodsA total of 46 healthy adults participated in studies exploring the biological variability of FLCs. Diurnal variation was investigated by collecting saliva samples immediately upon waking, 0.5 h, 3 h, 6 h, 9 h and 14 h post-waking. Saliva samples were assessed for FLCs, IgA, cortisol and dehydroepiandrosterone (DHEA). Between-day variation in FLCs and IgA was assessed by collecting saliva samples immediately upon waking for seven consecutive days. Participants underwent a dental examination to exclude oral health as a potential confounding variable. Within and between-person day-to day variation was explored in relation to a range of different factors: awakening time, sleep, exercise, well-being and alcohol consumption.ResultsSalivary secretion rates of FLCs decreased following waking and up to 3 h post-waking and then plateaued. This same pattern was observed for IgA. DHEA was stable upon waking and higher levels were seen in the morning with significantly lower levels thereafter. Cortisol levels significantly increased 0.5 h post-waking then continued to decline across the day. FLCs were significantly correlated with IgA but not cortisol or DHEA. Both FLCs and IgA parameters showed day-to-day variability, with coefficients of variation ≥ 40%. Earlier waking time was significantly correlated with higher FLC and IgA secretion rates. Inter-person differences in saliva parameter variability were observed but the degree of variation in FLCs and IgA was related within person. Inter-person day-to-day variation appeared to be uninfluenced by lifestyle or behavioural factors.ConclusionsSaliva FLCs secretion exhibits diurnal fluctuation that mirrors IgA fluctuation. Findings strongly indicate salivary FLC secretion is orchestrated by local plasma cells. FLCs and IgA both showed notable variability day-to-day, which was similar within person and influenced by awakening time. FLCs offer a promising adjunct to IgA in the measurement of oral immune activation

Investigating the utility of saliva immunoglobulins for the detection of myeloma and using myeloma proteins to clarify partition between oral and systemic immunity

OBJECTIVES Myeloma is characterised by the presence of monoclonal immunoglobulin (M-protein) and the free light chain (FLC) in blood. We investigated whether these M-proteins and FLC are detectable in myeloma patients' saliva to evaluate its utility for non-invasive screening and monitoring of haematological malignancies. METHODS A total of 57 patients with monoclonal gammopathy and 26 age-matched healthy participants provided paired serum and saliva samples for immunoglobulin characterisation and quantification. RESULTS Myeloma patients had IgG or IgA M-protein levels ranging up to five times and FLC levels up to a thousand times normal levels of polyclonal immunoglobulins. Despite these highly elevated levels, only two IgG and no IgA M-proteins or FLC could be detected in paired saliva samples. Most patients had reduced levels of serum polyclonal immunoglobulins, but all had normal levels of salivary IgA. CONCLUSIONS Immunoglobulin transfer from blood is not determined by levels in the systemic circulation and more likely dictated by periodontal inflammation and the integrity of the oral epithelium. Immunoglobulins secreted by bone marrow plasma cells do not substantially enter saliva, which represents a poor medium for myeloma diagnosis. These findings, along with normal salivary IgA levels despite systemic immunoparesis, support a strong partitioning of oral from systemic humoral immunity

Disordered Microbial Communities in the Upper Respiratory Tract of Cigarette Smokers

Cigarette smokers have an increased risk of infectious diseases involving the respiratory tract. Some effects of smoking on specific respiratory tract bacteria have been described, but the consequences for global airway microbial community composition have not been determined. Here, we used culture-independent high-density sequencing to analyze the microbiota from the right and left nasopharynx and oropharynx of 29 smoking and 33 nonsmoking healthy asymptomatic adults to assess microbial composition and effects of cigarette smoking. Bacterial communities were profiled using 454 pyrosequencing of 16S sequence tags (803,391 total reads), aligned to 16S rRNA databases, and communities compared using the UniFrac distance metric. A Random Forest machine-learning algorithm was used to predict smoking status and identify taxa that best distinguished between smokers and nonsmokers. Community composition was primarily determined by airway site, with individuals exhibiting minimal side-of-body or temporal variation. Within airway habitats, microbiota from smokers were significantly more diverse than nonsmokers and clustered separately. The distributions of several genera were systematically altered by smoking in both the oro- and nasopharynx, and there was an enrichment of anaerobic lineages associated with periodontal disease in the oropharynx. These results indicate that distinct regions of the human upper respiratory tract contain characteristic microbial communities that exhibit disordered patterns in cigarette smokers, both in individual components and global structure, which may contribute to the prevalence of respiratory tract complications in this population

Investigating the utility of saliva immunoglobulins for the detection of myeloma and using myeloma proteins to clarify partition between oral and systemic immunity

Objectives: Myeloma is characterised by the presence of monoclonal immunoglobulin (M-protein) and the free light chain (FLC) in blood. We investigated whether these M-proteins and FLC are detectable in myeloma patients’ saliva to evaluate its utility for non-invasive screening and monitoring of haematological malignancies. Methods: A total of 57 patients with monoclonal gammopathy and 26 age-matched healthy participants provided paired serum and saliva samples for immunoglobulin characterisation and quantification. Results: Myeloma patients had IgG or IgA M-protein levels ranging up to five times and FLC levels up to a thousand times normal levels of polyclonal immunoglobulins. Despite these highly elevated levels, only two IgG and no IgA M-proteins or FLC could be detected in paired saliva samples. Most patients had reduced levels of serum polyclonal immunoglobulins, but all had normal levels of salivary IgA. Conclusions: Immunoglobulin transfer from blood is not determined by levels in the systemic circulation and more likely dictated by periodontal inflammation and the integrity of the oral epithelium. Immunoglobulins secreted by bone marrow plasma cells do not substantially enter saliva, which represents a poor medium for myeloma diagnosis. These findings, along with normal salivary IgA levels despite systemic immunoparesis, support a strong partitioning of oral from systemic humoral immunity.</p

Assessing Bacterial Populations in the Lung by Replicate Analysis of Samples from the Upper and Lower Respiratory Tracts

<div><p>Microbes of the human respiratory tract are important in health and disease, but accurate sampling of the lung presents challenges. Lung microbes are commonly sampled by bronchoscopy, but to acquire samples the bronchoscope must pass through the upper respiratory tract, which is rich in microbes. Here we present methods to identify authentic lung microbiota in bronchoalveolar lavage (BAL) fluid that contains substantial oropharyngeal admixture. We studied clinical BAL samples from six selected subjects with potential heavy lung colonization. A single sample of BAL fluid was obtained from each subject along with contemporaneous oral wash (OW) to sample the oropharynx, and then DNA was extracted from three separate aliquots of each. Bacterial 16S rDNA sequences were amplified and products analyzed by 454 pyrosequencing. By comparing replicates, we were able to specify the depth of sequencing needed to reach a 95% chance of identifying a bacterial lineage of a given proportion—for example, at a depth of 5,000 tags, OTUs of proportion 0.3% or greater would be called with 95% confidence. We next constructed a single-sided outlier test that allowed lung-enriched organisms to be quantified against a background of oropharyngeal admixture, and assessed improvements available with replicate sequence analysis. This allowed identification of lineages enriched in lung in some BAL specimens. Finally, using samples from healthy volunteers collected at multiple sites in the upper respiratory tract, we show that OW provides a reasonable but not perfect surrogate for bacteria carried into to the lung by a bronchoscope. These methods allow identification of microbes that can replicate in the lung despite the background due to oropharyngeal microbes derived from aspiration and bronchoscopic carry-over.</p> </div

Comparison of reproducibility for all three BAL samples from subjects Tx 43 and Pulm 1.

<p>The subject is indicated at the top within each box, the replicates compared are indicated along each axis.</p

Characterization of lung-specific microorganisms using single sided outlier plots to compare single OW and BAL samples.

<p>A single OW sample (x-axis) and a single BAL sample (y-axis) were used for comparison for each subject (out of three samples of each for each subject). The color code indicates the results of significance testing for OTU outliers against a beta-binomial distribution. “FDR” indicates false discovery rate.</p