An ultrasensitive paper-based SERS sensor for detection of nucleolin using silver-nanostars, plastic antibodies and natural antibodies.

A state-of-the-art, ultrasensitive, paper-based SERS sensor has been developed using silver nanostars (AgNSs) in combination with synthetic and natural antibodies. A key component of this innovative sensor is the plastic antibody, which was synthesized using molecularly imprinted polymer (MIP) technology. This ground-breaking combination of paper substrates/MIPs with AgNSs, which is similar to a sandwich immunoassay, is used for the first time with the aim of SERS detection and specifically targets nucleolin (NCL), a cancer biomarker. The sensor device was carefully fabricated by synthesizing a polyacrylamide-based MIP on cellulose paper (Whatman Grade 1 filter) by photopolymerization. The binding of NCL to the MIP was then confirmed by natural antibody binding using a sandwich assay for quantitative SERS analysis. To facilitate the detection of NCL, antibodies were pre-bound to AgNSs with a Raman tag so that the SERS signal could indicate the presence of NCL. The composition of the sensory layers/materials was meticulously optimized. The intensity of the Raman signal at ∼1078 cm-1 showed a linear trend that correlated with increasing concentrations of NCL, ranging from 0.1 to 1000 nmol L-1, with a limit of detection down to 0.068 nmol L-1 in human serum. The selectivity of the sensor was confirmed by testing its analytical response in the presence of cystatin C and lysozyme. The paper-based SERS detection system for NCL is characterized by its simplicity, sustainability, high sensitivity and stability and thus embodies essential properties for point-of-care applications. This approach is promising for expansion to other biomarkers in various fields, depending on the availability of synthetic and natural antibodies.

Analysis of Natural Antibodies during the Development of Phantom Pain Syndrome.

We determined natural antibodies (n-Abs) to the regulators of the main systems of biochemical homeostasis: ß-endorphin, serotonin, dopamine, histamine, orphanin, angiotensin, GABA, glutamate, bradykinin, vasopressin, thrombin, and α-2-macroglobulin in individuals with phantom pain syndrome (PPS), resulting from amputation after injury. It was established that each patient has an individual immunoprofile, but for all of them there was a significant increase in the level of antibodies to serotonin, histamine, and angiotensin, which reflect the chronicity of the pain syndrome and do not depend on the self-assessment of the severity of PPS. Determination of the role of regulators of biochemical homeostasis in the development of phantom pain showed that, at high, moderate, and weak severity of PPS, the biogenic amine and angiotensinergic systems are activated. A decrease in PPS intensity normalizes deviations in all immunological parameters. The levels of n-Abs for the pain (ß-endorphin) and analgesic (orphanin) systems are significant only at low PPS. Monitoring the individual profile of n-Abs to endogenous regulators allows us to obtain an objective picture of the pain status of the patient's body.

Loss of TET2 increases B-1 cell number and IgM production while limiting CDR3 diversity.

Recent studies have demonstrated a role for Ten-Eleven Translocation-2 (TET2), an epigenetic modulator, in regulating germinal center formation and plasma cell differentiation in B-2 cells, yet the role of TET2 in regulating B-1 cells is largely unknown. Here, B-1 cell subset numbers, IgM production, and gene expression were analyzed in mice with global knockout of TET2 compared to wildtype (WT) controls. Results revealed that TET2-KO mice had elevated numbers of B-1a and B-1b cells in their primary niche, the peritoneal cavity, as well as in the bone marrow (B-1a) and spleen (B-1b). Consistent with this finding, circulating IgM, but not IgG, was elevated in TET2-KO mice compared to WT. Analysis of bulk RNASeq of sort purified peritoneal B-1a and B-1b cells revealed reduced expression of heavy and light chain immunoglobulin genes, predominantly in B-1a cells from TET2-KO mice compared to WT controls. As expected, the expression of IgM transcripts was the most abundant isotype in B-1 cells. Yet, only in B-1a cells there was a significant increase in the proportion of IgM transcripts in TET2-KO mice compared to WT. Analysis of the CDR3 of the BCR revealed an increased abundance of replicated CDR3 sequences in B-1 cells from TET2-KO mice, which was more clearly pronounced in B-1a compared to B-1b cells. V-D-J usage and circos plot analysis of V-J combinations showed enhanced usage of VH11 and VH12 pairings. Taken together, our study is the first to demonstrate that global loss of TET2 increases B-1 cell number and IgM production and reduces CDR3 diversity, which could impact many biological processes and disease states that are regulated by IgM.

Natural antibodies targeting LPS in pleural effusions of various etiologies.

Respiratory infection, cancer, and heart failure can cause abnormal accumulation of fluid in the pleural cavity. The immune responses within the cavity are orchestrated by leucocytes that reside in the serosal-associated lymphoid tissue. Natural antibodies (NAbs) are abundant in the serum (S) having a major role in systemic and mucosal immunity; however, their occurrence in pleural fluid (PF) remains an open question. Our aim herein was to detect and measure the levels of NAbs (IgM, IgG, IgA) targeting lipopolysaccharides (LPS) in both the pleural fluid and the serum of 78 patients with pleural effusions (PEs) of various etiologies. The values of anti-LPS NAb activity were extracted through a normalization step regarding the total IgM, IgG, and IgA levels, all determined by in-house ELISA. In addition, the ratios of PF/S values were analyzed further with other critical biochemical parameters from pleural fluids. Anti-LPS NAbs of all Ig classes were detected in most of the samples, while a significant increase of anti-LPS activity was observed in infectious and noninfectious compared with malignant PEs. Multivariate linear regression confirmed a negative correlation of IgM and IgA anti-LPS PF/S ratio with malignancy. Moreover, anti-LPS NAbs PF/S measurements led to increased positive and negative predictive power in ROC curves generated for the discrimination between benign and malignant PEs. Our results highlight the role of anti-LPS NAbs in the pleural cavity and demonstrate the potential translational impact that should be further explored.NEW & NOTEWORTHY Here we describe the detection and quantification of natural antibodies (NAbs) in the human pleural cavity. We show for the first time that IgM, IgG, and IgA anti-LPS natural antibodies are detected and measured in pleural effusions of infectious, noninfectious, and malignant etiologies and provide clinical correlates to demonstrate the translational impact of our findings.

The role of B-1 cells in cancer progression and anti-tumor immunity.

In recent years, in addition to the well-established role of T cells in controlling or promoting tumor growth, a new wave of research has demonstrated the active involvement of B cells in tumor immunity. B-cell subsets with distinct phenotypes and functions play various roles in tumor progression. Plasma cells and activated B cells have been linked to improved clinical outcomes in several types of cancer, whereas regulatory B cells have been associated with disease progression. However, we are only beginning to understand the role of a particular innate subset of B cells, referred to as B-1 cells, in cancer. Here, we summarize the characteristics of B-1 cells and review their ability to infiltrate tumors. We also describe the potential mechanisms through which B-1 cells suppress anti-tumor immune responses and promote tumor progression. Additionally, we highlight recent studies on the protective anti-tumor function of B-1 cells in both mouse models and humans. Understanding the functions of B-1 cells in tumor immunity could pave the way for designing more effective cancer immunotherapies.

Influence of microbiota-driven natural antibodies on dengue transmission.

Dengue has had a significant global health impact, with a dramatic increase in incidence over the past 50 years, affecting more than 100 countries. The absence of a specific treatment or widely applicable vaccine emphasizes the urgent need for innovative strategies. This perspective reevaluates current evidence supporting the concept of dual protection against the dengue virus (DENV) through natural antibodies (NAbs), particularly anti-α-Gal antibodies induced by the host's gut microbiome (GM). These anti-α-Gal antibodies serve a dual purpose. Firstly, they can directly identify DENV, as mosquito-derived viral particles have been observed to carry α-Gal, thereby providing a safeguard against human infections. Secondly, they possess the potential to impede virus development in the vector by interacting with the vector's microbiome and triggering infection-refractory states. The intricate interplay between human GM and NAbs on one side and DENV and vector microbiome on the other suggests a novel approach, using NAbs to directly target DENV and simultaneously disrupt vector microbiome to decrease pathogen transmission and vector competence, thereby blocking DENV transmission cycles.

Natural antibodies are required for clearance of necrotic cells and recovery from acute liver injury.

Background & Aims: Hepatocellular necrosis is common in both acute and chronic liver injury and may evolve to fibrosis and liver failure. Injury leads to accumulation of necrotic cell debris in the liver, which drives persistent inflammation and poor recovery. This study investigated the role of natural antibodies (NAbs) in the clearance of necrotic cells in the injured liver, their impact on tissue regeneration and their potential as a therapy for acute liver injury. Methods: We used murine models of drug-induced liver injury and focal thermal injury in immunocompetent and antibody-deficient mice (Rag2-/- and IgMi). Intravital microscopy was used to investigate the role of NAbs in the phagocytosis of necrotic cells in the liver in vivo. Immunostainings were used to quantify the extent of liver necrosis (fibrin), antibody deposition (IgM and IgG) and cellular proliferation (Ki67). Results: Both IgM and IgG NAbs bound necrotic liver areas and opsonized multiple debris molecules released during hepatocellular necrosis such as DNA, histones, actin, phosphoinositides and mitochondrial cardiolipin, but not phosphatidylserine. Rag2-/- and IgMi mice presented impaired recovery from liver injury, which was correlated to the sustained presence of necrotic debris in the tissue, prolonged inflammation and reduced hepatocellular proliferation. These defects were rescued by treating mice with NAbs after the induction of injury. Mechanistically, in vitro and in vivo, phagocytosis of necrotic debris was dependent on NAbs via Fcγ receptors and CD11b. Moreover, NAb-mediated phagocytosis of necrotic cell debris occurs in two waves, firstly driven by neutrophils and then by recruited monocytes. Importantly, supplementation of immunocompetent mice with NAbs also improved liver regeneration significantly, demonstrating the therapeutic potential of natural IgM and IgG. Conclusion: NAbs drive the phagocytosis of necrotic cells in liver injury and promote liver regeneration and recovery. Impact and implications: Treatment with natural antibodies after acute liver injury improved recovery by increasing the clearance of necrotic debris and by improving cellular proliferation in the liver. This preclinical study provides a basis for the development of an immunotherapy for patients with early-stage, reversible, liver injury that aims to prevent disease chronification into fibrosis and liver failure.

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.

Deficiency in the production of antibodies to lipids correlates with increased lipid metabolism in severe COVID-19 patients.

Background: Antibodies to lipids are part of the first line of defense against microorganisms and regulate the pro/anti-inflammatory balance. Viruses modulate cellular lipid metabolism to enhance their replication, and some of these metabolites are proinflammatory. We hypothesized that antibodies to lipids would play a main role of in the defense against SARS-CoV-2 and thus, they would also avoid the hyperinflammation, a main problem in severe condition patients. Methods: Serum samples from COVID-19 patients with mild and severe course, and control group were included. IgG and IgM to different glycerophospholipids and sphingolipids were analyzed using a high-sensitive ELISA developed in our laboratory. A lipidomic approach for studying lipid metabolism was performed using ultra-high performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight mass spectrometry (UHPLC-ESI-QTOF-MS). Results: Mild and severe COVID-19 patients had higher levels of IgM to glycerophosphocholines than control group. Mild COVID-19 patients showed higher levels of IgM to glycerophosphoinositol, glycerophosphoserine and sulfatides than control group and mild cases. 82.5% of mild COVID-19 patients showed IgM to glycerophosphoinositol or glycerophosphocholines plus sulfatides or glycerophosphoserines. Only 35% of severe cases and 27.5% of control group were positive for IgM to these lipids. Lipidomic analysis identify a total of 196 lipids, including 172 glycerophospholipids and 24 sphingomyelins. Increased levels of lipid subclasses belonging to lysoglycerophospholipids, ether and/or vinyl-ether-linked glycerophospholipids, and sphingomyelins were observed in severe COVID-19 patients, when compared with those of mild cases and control group. Conclusion: Antibodies to lipids are essential for defense against SARS-CoV-2. Patients with low levels of anti-lipid antibodies have an elevated inflammatory response mediated by lysoglycerophospholipids. These findings provide novel prognostic biomarkers and therapeutic targets.

Antibody-Dependent Enhancement with a Focus on SARS-CoV-2 and Anti-Glycan Antibodies.

Antibody-dependent enhancement (ADE) is a phenomenon where virus-specific antibodies paradoxically cause enhanced viral replication and/or excessive immune responses, leading to infection exacerbation, tissue damage, and multiple organ failure. ADE has been observed in many viral infections and is supposed to complicate the course of COVID-19. However, the evidence is insufficient. Since no specific laboratory markers have been described, the prediction and confirmation of ADE are very challenging. The only possible predictor is the presence of already existing (after previous infection) antibodies that can bind to viral epitopes and promote the disease enhancement. At the same time, the virus-specific antibodies are also a part of immune response against a pathogen. These opposite effects of antibodies make ADE research controversial. The assignment of immunoglobulins to ADE-associated or virus neutralizing is based on their affinity, avidity, and content in blood. However, these criteria are not clearly defined. Another debatable issue (rather terminological, but no less important) is that in most publications about ADE, all immunoglobulins produced by the immune system against pathogens are qualified as pre-existing antibodies, thus ignoring the conventional use of this term for natural antibodies produced without any stimulation by pathogens. Anti-glycan antibodies (AGA) make up a significant part of the natural immunoglobulins pool, and there is some evidence of their antiviral effect, particularly in COVID-19. AGA have been shown to be involved in ADE in bacterial infections, but their role in the development of ADE in viral infections has not been studied. This review focuses on pros and cons for AGA as an ADE trigger. We also present the results of our pilot studies, suggesting that AGAs, which bind to complex epitopes (glycan plus something else in tight proximity), may be involved in the development of the ADE phenomenon.

Comparative Determination of Natural Antibodies for the Assessment of the Cardiovascular System and the Level of Human Difficulty.

A comparative analysis of natural antibodies to ß-endorphin, angiotensin, dopamine, serotonin, parameters of the cardiovascular system and anxiety levels was carried out for 241 athletes of various qualifications and sports. The obtained indicators of the cardiovascular system were compared with reference values. A significant increase in the level of natural antibodies to angiotensin was established for all groups of athletes. In the case of dopamine, serotonin, these differences are associated with the qualification of the athlete, for ß-endorphin, differences in the level of the indicator depending on the sport were found. A group of individuals with high levels of situational and personal anxiety was found among highly qualified athletes. An increase in blood pressure in athletes of cyclic sports and martial arts is adaptive, and in athletes of speed-strength sports it leads to a change in the walls of the myocardium. As a result of the study, the possibility of a comprehensive determination of natural antibodies and functional indicators as diagnostic markers for assessing the state of the human cardiovascular system has been shown.

Natural autoimmunity in oligoarticular juvenile idiopathic arthritis.

BACKGROUND: Oligoarticular juvenile idiopathic arthritis (oligo-JIA) is considered as an antigen-driven lymphocyte-mediated autoimmune disease. Natural antibodies (NAbs) are pre-immune antibodies produced in the absence of exogenous antigen stimulation, participating in both, innate and adaptive immunity. Considering their major immunoregulatory role in homeostasis and autoimmune pathogenesis, we designed this study to further elucidate their role in oligo-JIA pathogenesis. METHODS: Seventy children with persistent oligo-JIA and 20 healthy matched controls were enrolled in the study. Serum IgM and IgA antibodies against human G-actin, human IgG F(ab΄)2 fragments and the hapten TriNitroPhenol (TNP) as well as the total concentration of serum IgM and IgA were measured by in-house enzyme-immunoassays. Kolmogorov-Smirnov normality test, Kruskal-Wallis H and Mann-Whitney tests were used to assess data distribution, and significant differences of non-parametric data between groups of the study. Backward regression analysis was used to analyze the effect of multiple factors (age, gender, disease activity, anti-nuclear antibody positivity, presence of uveitis) on continuous dependent variables (activities and activity/ concentration ratios of IgM and IgA NAbs). RESULTS: The ratios of IgA anti-TNP, anti-actin and anti-F(ab΄)2 levels to total serum IgA concentration were found to be significantly increased in patients with oligo-JIA compared to healthy subjects. Significantly elevated levels of IgM anti-TNP antibodies were also found in children with inactive oligo-JIA compared to those of children with active disease and of healthy controls. In the presence of anterior uveitis, IgM anti-TNP levels were significantly higher than in patients without uveitis or in healthy controls. Backward regression analysis revealed that the disease activity and the presence of anterior uveitis independently affect IgM anti-TNP levels. CONCLUSUIONS: Our findings are in accordance with the hypothesis that NAbs contribute to the pathogenesis of autoimmune diseases and provide additional evidence that disturbances in natural autoimmunity may contribute to the as yet unclarified pathogenesis of oligo-JIA.

The characters of antibodies against PLA2R in healthy individuals and in the patient with PLA2R associated membranous nephropathy.

BACKGROUND: Most primary membranous nephropathy (MN) is mediated by anti-phospholipase A2 receptor (PLA2R) antibodies. Recently, these antibodies have been revealed months to years before the disease's onset. Their production and pathogenicity need further investigation. METHODS: Anti-PLA2R antibodies were purified from plasma of eight healthy individuals, 12 patients with PLA2R-related MN and negative circulating antibody (Ab-), and 18 patients with positive anti-PLA2R antibodies (Ab +), using affinity column coupled with recombinant human PLA2R. The antigen specificity, antibody amount, titer, IgG subclass, and affinity were assessed by Western blot, immunofluorescence, ELISA, and surface plasmon resonance. RESULTS: The natural anti-PLA2R antibodies recognized the conformational structure of PLA2R which locates on the cell membrane of podocytes. The amount of natural IgG was 0.12 ± 0.04 g/L, which accounted for 0.80% of total IgG and was lower than that of patients (2.36%, P < 0.001). The titer of natural antibodies was lower than that of patients in Ab- and Ab + groups (1:16 vs. 1:43 vs. 1:274, P < 0.001). IgG2(45.1%) was predominant in natural antibodies, while IgG4 was predominant in Ab + group (45.7 vs. 25.0%, P < 0.001). IgG1 was increasing from natural antibodies to Ab- and Ab + groups. The affinity of natural antibodies was lower than that of patients (KD: 641.0 vs. 269.0 vs. 99.6 nM, P = 0.002). The antibody titer, affinity, and IgG4 percentage were associated with the severity of proteinuria and the stages of membranous lesion. CONCLUSIONS: The natural anti-PLA2R antibodies exist in healthy plasma. The antibody titer, IgG subclass, and affinity may participate in the pathogenesis of anti-PLA2R antibodies.

Decrease in naturally occurring antibodies against epitopes of Alzheimer's disease (AD) risk gene products is associated with cognitive decline in AD.

BACKGROUND: Naturally occurring antibodies (NAbs) are germline-encoded immunoglobulins that can bind to and clear out self-neo-epitopes as well as apoptotic and necrotic cells. However, NAbs pathological relevance in Alzheimer's disease (AD) is not well-understood. METHODS: Twenty-eight candidate proteins encoded by AD-associated genes were selected for this study based on a number of selection criteria, including preferential expression in the brain and B-lymphocyte cells. The levels of NAbs in plasma were analyzed according to their epitopes in age- and gender-matched cognitively normal subjects (CN, n = 56), subjects with mild cognitive impairment (MCI, n = 16) and subjects with AD (n = 56). We aimed to study the levels of their NAbs in plasma and their associations with cognitive decline in individuals with AD. RESULTS: Of the 28 antigens tested, 17 showed decreased NAbs in individuals with AD; in particular, NAb-TREM2 had an area under the ROC curve of 0.806, with the highest sensitivity (0.370) at 95% specificity among all 28 tests. Further protein-protein interaction networks and functional enrichment analysis suggested that target genes were enriched in AD-related pathological processes classified under "Alzheimer's disease", "neurodegenerative disease" and "amyloidosis". The "Alzheimer's disease" and "neurodegenerative disease" clusters, which converged on the initial "recognition" step of microglial phagocytosis, showed the best diagnostic performance for AD. CONCLUSIONS: This study suggests a decline in the function of the adaptive immune system in AD, and the levels of circulating NAbs are likely to serve as biomarkers for surveilling the progression of AD.

Analysis of Immunobiochemical Parameters in Overweight People in Assessing the Risk of Cardiovascular Diseases.

A comparative analysis of specific immunobiochemical parameters, including natural antibodies (NAb) to endogenous regulators of the cardiovascular system, adrenal and gastrointestinal hormones, was performed in students aged 18-22 years with normal and increased body weight (the body mass index from 18.5 to 24.9 kg/m2 and from 25 to 29.9 kg/m2, respectively). The serum content of NAb and hormones was determined by ELISA. The level of the studied indicators depended on the body mass index value. In overweight subjects, the main immune indicators of the biogenic amine system, renin-angiotensin system, and kinin system exceeded the normal. The cortisol level was higher than in subjects with normal body weight. Aldosterone secretion was less dependent on the ACTH content and was lower than in students with normal body weight. The content of cholecystokinin and gastrin corresponded to the values for overweight. These trends in hormone contents are a predisposing factor for further weight gain. Practical significance of the combined assessment of disturbances in the immunological and biochemical homeostasis has been established. Analysis of the adrenal and gastrointestinal hormones can predict the risk of weight gain, but at the same time, changes in the level of immunological indicators in subjects with increased body weight characterizes the possibility of developing cardiovascular pathologies.

Natural Antibodies Mediate Protection Against Acinetobacter baumannii Respiratory Infections.

BACKGROUND: Acinetobacter baumannii causes a wide range of dangerous infections due to the emergence of pandrug-resistant strains. Therefore, there is a need for alternative therapeutics to treat these infections, including those targeting the host immune responses. However, immune responses, especially the humoral response against this pathogen, are poorly understood. METHODS: This study investigated the lymphocyte-mediated innate immune resistance to A. baumannii AB5075 pulmonary infection using B- and T-cell-deficient (Rag2-/-) mice, the protective effect of natural antibodies (NAbs), and the expression of complement-mediated responses using a mouse pneumonia model. RESULTS: Our results showed that intranasally infected Rag2-/- mice are impaired in clearing bacteria from lung, liver, and spleen at 24 hours postinfection compared to wildtype mice. Animal pretreatment with normal mouse serum or purified antibodies from naive mice rescued Rag2-/- mice from infection. Analysis of C3 complement protein binding demonstrated that NAbs increased C3 protein deposition on A. baumannii cells, indicating the activation of the classical complement pathway by NAbs. CONCLUSIONS: Overall, our study shows that NAbs mediate innate immune resistance against A. baumannii, a finding that may lead to the development of effective therapies against human infections caused by this antibiotic-resistant A. baumannii.

Levels of Circulating IgM and IgY Natural Antibodies in Broiler Chicks: Association with Genotype and Farming Systems.

Naturally occurring antibodies (NAbs), which are major components of innate immunity, exist in circulation under healthy conditions without prior antigenic stimulation and are able to recognize both self- and non-self-constituents. The present study aimed at identifying potential immunological differences between commercial fast- and slow-growth broilers (n = 555) raised in conventional and free-range systems, respectively, through the use of the specificity, isotypes and levels of circulating NAbs. The possible beneficial effect of oregano-based dietary supplementation was also evaluated. To this end, serum IgM and IgY NAbs against self- (actin and DNA) and non-self- antigens (trinitrophenol and lipopolysaccharide) were measured by ELISA and further correlated with genotype, season and performance. Significantly higher levels of IgM NAbs against all antigens were found in slow-growth compared to fast-growth broilers. IgM NAb levels were also significantly increased in dietarily supplemented slow-growth broilers versus those consuming standard feed. Moreover, significantly elevated levels of anti-DNA IgY NAbs were found in fast-growth compared to slow-growth broilers, whereas the opposite was observed for anti-LPS IgY NAbs. Multivariate linear regression analysis confirmed multiple interactions between NAb levels, genotype, season and performance. Overall, serum NAbs have proven to be valuable innovative immunotools in the poultry industry, efficiently differentiating fast-growing versus slow-growing broilers, and dietary supplementation of plant extracts can enhance natural immunity.

Further analysis of natural antibodies against ischemic stroke.

Background: Our previous study revealed that circulating levels of IgG natural antibodies (NAbs) for vascular endothelial growth factor receptor 1 (VEGFR1) were significantly decreased in patients with arteriosclerosis compared with control subjects. To enhance the sensitivity of an enzyme-linked immunosorbent assay (ELISA) developed in-house for antibody testing, the present work was designed to investigate additive signals in the in-house ELISA developed with the combination of two or more linear peptide antigens derived from target proteins of interest, including VEGFR1, oxidized low-density lipoprotein receptor 1 (LOX-1), interleukins 6 (IL6) and 8 (IL8). Methods: A total of 218 patients with ischemic stroke and 198 healthy controls were enrolled and an in-house ELISA was developed with linear peptides derived from VEGFR1, LOX-1, IL6, and IL8 to detect their IgG levels in plasma. Results: Compared with control subjects, plasma levels of IgG NAbs for the IL6-IL8 combination were significantly lower in female patients (Z = -3.149, P = 0.002), whereas male patients showed significantly lower levels of plasma anti-VEGFR IgG (Z = -3.895, P < 0.001) and anti-LOX1a IgG (Z = -4.329, P < 0.001). Because plasma levels of IgG NAbs for both the IL6-IL8-LOX1a-LOX1b combination and the VEGFR1a-VEGFR1b-LOX1a-LOX1b combination were significantly lower in the patient group than the control group, receiver operating characteristic (ROC) analysis was performed and the results showed that the VEGFR1a-VEGFR1b-LOX1a-LOX1b combination had an area under the ROC curve (AUC) of 0.70 for its IgG assay with a sensitivity of 27.1% against the specificity of 95.5% and that the IL6-IL8-LOX1a-LOX1b combination had an AUC of 0.67 for its IgG assay with a sensitivity of 21.1% against the specificity of 95.5%. Spearman correlation analysis showed that plasma IgG NAbs against the IL6-IL8 combination were positively correlated with carotid plaque size only in male patients (r = 0.270, p = 0.002). Conclusions: Circulating IgG NAbs for the target molecules studied may be potential biomarkers for a subgroup of ischemic stroke and also contribute to the gender differences in clinical presentation of the disease.

Integrative assessment of immunity, health-state, growth and survival of Magellanic penguin chicks in a colony exposed to ecotourism.

Accumulating reports of negative impacts of tourist activities on wildlife emphasize the importance of closely monitoring focal populations. Although some effects are readily noticed, more subtle ones such as changes in physiological functions of individuals might go overlooked. Based on evidence of altered physiology associated with ecotourism on Magellanic penguins Spheniscus magellanicus, here we performed an integrated assessment using a diverse physiological toolkit together with more traditional fitness-related measures to better understand mechanisms and potential consequences. Chicks exposed to tourism showed altered immune parameters and elevated flea prevalence, reinforcing previous findings. Tourism-exposed female, but not male, chicks also showed relatively lower hematocrit and plasma protein levels, providing evidence consistent with a sex-specific response to tourist visitation. Physiological alterations detected in tourism-exposed young chicks (week 1-2) were maintained and the effect on flea infestation increased during the study period (week 4-5 of post-hatch). Despite the effects on physiology, these did not seem to translate into immediate fitness costs. No detectable tourism effects were found on brood sex ratios, chick growth and body condition, and survival until week 5-6 post-hatch. We detected no effects on reproductive output and only a marginal effect on nest survival during incubation despite previous reports of tourism-associated alterations in stress indices of adults. This disconnection could result if the physiological changes are not strong enough to impact fitness, if effects balance each other out, or if changes are part of a copying strategy. Alternatively, the physiological alterations might only show impacts later in the brooding cycle or even after chick emancipation from their parents. Our results suggest that integrative monitoring of potential anthropogenic impacts on wildlife should include evaluation of physiological mechanisms and individual-level responses in populations exposed to human activities.

TNFRSF13B in B cell responses to organ transplantation.

Antibodies directed against organ transplants are thought to pose the most vexing hurdle to enduring function and survival of the transplants, particularly organ xenotransplants, and accordingly basic and clinical investigation has focused on elucidating the specificity and pathogenicity of graft-specific antibodies. While much has been learned about these matters, far less is known about the B cells producing graft-specific antibodies and why these antibodies appear to injure some grafts but not others. With the goal of addressing those questions, we have investigated the properties of tumor necrosis factor receptor super family-13B (TNFRSF13B), which regulates various aspects of B cell responses. A full understanding of the functions of TNFRSF13B however is hindered by extreme polymorphism and by diversity of interactions of the protein. Nevertheless, TNFRSF13B variants have been found to exert distinct impact on natural and elicited antibody responses and host defense and mutations of TNFRSF13B have been found to influence the propensity for development of antibody-mediated rejection of organ transplants. Because B cell responses potentially limit application of xenotransplantation, understanding how TNFRSF13B diversity and TNFRSF13B variants govern immunity in xenotransplantation could inspire development of novel therapeutics that could in turn accelerate clinical implementation of xenotransplantation.