Anti-lipid Antibodies Research Articles

8150 CD1a Lipid Presentation Contributes to T2D Phenotype in DIO Mouse Model

Abstract Disclosure: J.A. Ayala Angulo: None. K.D. Wiggins: None. M. Lopez: None. D. Nicholas: None. Type 2 Diabetes’s complex etiology, progression, and comorbidities are not fully explained by current models. Elevation of inflammatory markers and immune cell infiltration into tissues of T2D patients has been described in the literature extensively, but no clear answers as to what antigens are driving this response and how these observations contribute to disease progression exist. Recently, CD1a has been shown to present lipids to T cells and induce a memory response. Our previous work shows that T2D patients produce anti-lipid antibodies likely derived with T cell help, not present in healthy patients. Together, these data suggest a potential role for lipid-specific T cell responses to drive T2D progression through inflammatory stress, antibody production, and tissue infiltration. Current work with human CD1a transgenic B6 mice points to elevated liver steatosis and hepatitis along with higher glucose levels relative to low fat diet controls. CD1a mice show smaller islets suggesting pancreatic atrophy. This validates the CD1a-T cell mechanism as a potential contributor to T2D but also introduces a more physiologically relevant mouse model of T2D. Presentation: 6/1/2024

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

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. 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). 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. 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.

Decrease of glutathione peroxidase in arrhythmic cardiac pathology in young individuals and its therapeutic implications.

Glutathione peroxidase (GPx), as an antioxidant enzyme, is involved in the regulation of processes that cause cellular oxidative stress, with implications in various pathologies. The aim of the present study was to evaluate GPx variations in patients with arrhythmic, non-structural cardiac disorders. The research was performed on 120 patients, with a mean age of 33 years old, divided into 3 equal groups, of which 2 groups included patients with cardiac arrhythmias, the first group, associated with dyslipidemia and the second one, without dyslipidemia, and a control group consisting of healthy individuals. The method for determining GPx was based on the GPx enzyme catalysis reaction of the reduced glutathione (GSH) oxidation reaction by cumene hydroperoxide. The results revealed that GPx variation was decreased in patients with cardiac arrhythmias, with or without dyslipidemia, up to 66 and 74% of mean control values, respectively, the differences being statistically significant, showing the existence of an oxidative stress imbalance, that may be involved in triggering arrhythmogenic electrochemical mechanisms. The GPx deficiency determined in relation to cardiac arrhythmias was in dyslipidemic and non-lipidemic patients as follows: 29-35% in sinus bradycardia, 31-35% in associated cardiac arrhythmias, 30-33% in sinus tachycardia, 27-33% in atrial fibrillation, 32-33% in atrial flutter, 27-32% in atrial extrasystolic arrhythmia, 28-30% in ventricular extrasystolic arrhythmia and 18-26% in paroxysmal supraventricular tachycardia. Collectively, the results revealed that GPx, an antioxidant enzyme, is a specific biomarker, whose decrease indicated the existence of oxidative stress in young individuals with cardiac arrhythmias and its involvement in arrhythmogenic electrochemical processes. In addition, GPx deficiencies were between 18-35% in all types of cardiac arrhythmias, the highest being recorded in sinus bradycardia and the lowest in paroxysmal supraventircular tachycardia. Furthermore, the oxidative stress favored by the decrease of GPx induced lipid oxidation, regardless of the presence or absence of dyslipidemia, which triggered the formation of anti-lipid antibodies and a subclinical endothelial aggression, with early atherosclerotic potential. GPx evaluation may argue for the existence of oxidative stress in non-structural cardiac arrhythmias, and by its proper correction (antioxidants), prophylaxis of atherogenic dysfunction.

Liposomes Bearing Non-Bilayer Phospholipid Arrangements Induce Specific IgG Anti-Lipid Antibodies by Activating NK1.1+, CD4+ T Cells in Mice.

Liposomes are artificial models of cellular membranes that are used as delivery systems for genes, drugs and protein antigens. We have previously used them to study the antigenic properties of their phospholipids. Here, we used them to induce the production of IgG anti-non-bilayer phospholipid arrangements (NPAs) antibodies in mice; these antibodies cause cell lysis and trigger a lupus-like disease in mice. We studied the mechanisms that lead to the production of these antibodies, and provide evidence that NK1.1+, CD4+ T cells respond to NPA-bearing liposomes and deliver the help required for specific B cell activation and antibody class-switching to IgG. We found increased numbers of IL-4-producing NK1.1+, CD4+ T cells in the secondary lymphoid organs of mice administered with NPAs, and these cells also expressed CD40L, which is required for B cell activation. Additionally, we isolated and purified NK1.1+, CD4+ T cells from spleens and determined that they over-expressed 40 genes, which are key players in inflammatory processes and B cell stimulation and have TRAF6 and UNC39B1 as key nodes in their network. These results show that liposomes are membrane models that can be used to analyze the immunogenicity of lipids.

Use of host lipids by the Lyme disease spirochete may lead to biomarkers.

Lyme disease is the most common tick-borne disease in North America and Europe, however, current biomarkers inconsistently detect the disease. In this issue of the JCI, Gwynne et al. revealed how the Lyme disease agent Borrelia burgdorferi relies on host lipids for growth. The authors used a murine model to show that B. burgdorferi infection led to the production of antibodies against phospholipids, possibly as a consequence of incorporation into the spirochete membrane. Antibodies were induced against phosphatidic acid, phosphatidylcholine, and phosphatidylserine. Notably, no antibodies against cardiolipin were found, distinguishing Lyme disease from syphilis and some other diseases. Sera samples from patients with Lyme disease suggested that these antibodies may help diagnose B. burgdorferi infection and that antibody titers may effectively indicate the response to treatment. These findings suggest that B. burgdorferi–induced anti-lipid antibodies, in conjunction with a careful clinical assessment, may aid in the diagnosis of Lyme disease.

Study of specific plasmablasts and plasma cells to lipidic particles in the mouse models of lupus generated by liposomes bearing lipidic particles stabilized with different inducers

Abstract Systemic lupus erythematosus (SLE) is an inflammatory, multisystem, chronic an autoimmune disease of unknown etiology. In our research group, we developed a mouse model of lupus by the administration of liposomes bearing lipid particles stabilized by chlorpromazine, promazine or manganese in BALB/c mice. In this model, we have detected IgG antibodies against lipid particles, so we are studying the immune mechanisms of how IgG antibodies are generated against a lipid antigen. It is noteworthy that the production mechanisms of anti-lipid IgG antibodies are practically unknown and have not been studied in autoimmune diseases such as SLE. In previous works, we showed that B cells mainly respond through germinal center to generate IgG antibodies against lipid particles. Moreover, against protein antigens after germinal center reaction, B cells differentiate into secondary lymphoid organs to plasmablasts that are maintained in cell cycle, can be divided and are in circulation. Eventually these plasmablasts can differentiate into plasma cells that are no longer in the cell cycle and are set in a niche (mainly in bone marrow or less in secondary lymphoid organs) where they begin to secrete antibodies. In this work, we propose to study the mechanisms of plasmablasts and plasma cells, specific to a lipid antigen, that leads to the production of IgG antibodies. This is very important because the IgG autoantibodies are the main causes of autoimmunity and particularly the anti-lipid particles antibodies, which have been found in patients with SLE, leprosy and preeclampsia, are those that trigger the lupus like disease in the mouse.

Affinity maturation of anti-lipid IgG antibodies produced via germinal center

Abstract Non-bilayer phospholipid arrangements (NPA) are lipid associations different to the bilayer and are found on cell membranes; they participate in different cellular functions and are transient, but when they are stabilized by drugs such as chlorpromazine, they become immunogenic and induce the formation of anti-NPA antibodies. Mice that receive stabilized NPA share several characteristics with patients with systemic lupus erythematosus, such as the presence of anti-cardiolipin, anti-histone and anti-nuclear antibodies, lupus band, malar exanthema and glomerulonephritis. Mice with this lupus model have been shown to develop the disease as a consequence of the production of anti-NPA antibodies; in addition, most of these antibodies are of the IgG class and are produced mainly by the germinal center pathway. In the case of protein antigens, germinal centers are the sites where class-switch recombination, somatic mutation, memory generation and affinity maturation occur. To determine if affinity maturation, a hallmark of the antibodies produced via germinal center reactions, occurs in the anti-NPA IgG antibodies produced in germinal centers, we performed antigen-specific ELISAs, both in the absence and in the presence of urea. We observed that the percentage of urea-resistant (high affinity) anti-NPA IgG antibodies in relation to the total anti-NPA IgG antibodies increases over time, and it correlates with the number of NPA-specific germinal center B cells.

Corrigendum: Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus

[This corrects the article on p. 396 in vol. 7, PMID: 27746783.].

Lysosomal Storage Disorders and Malignancy.

Lysosomal storage disorders (LSDs) are infrequent to rare conditions caused by mutations that lead to a disruption in the usual sequential degradation of macromolecules or their transit within the cell. Gaucher disease (GD), a lipidosis, is among the most common LSD, with an estimated incidence of 1 in 40,000 among the Caucasian, non-Jewish population. Studies have indicated an increased frequency of polyclonal and monoclonal gammopathy among patients with GD. It has been shown that two major sphingolipids that accumulate in GD, namely, β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Investigations undertaken in an affected mouse model revealed βGL1-22- and LGL1-specific NKT cells were present and constitutively promoted the expression of a T-follicular helper (TFH) phenotype; injection of these lipids led to downstream induction of germinal center B cells, hypergammaglobulinemia, and the production of antilipid antibodies. Subsequent studies have found clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC). Furthermore, substrate reduction ameliorated GD-associated gammopathy in mice. It had been hypothesized that chronic antigenic stimulation by the abnormal lipid storage and associated immune dysregulation may be the underlying mechanism for the increased incidence of monoclonal and polyclonal gammopathies, as well as an increased incidence of multiple myeloma in patients with GD. Current observations support this proposition and illustrate the value of investigations into rare diseases, which as ‘experiments of nature’ may provide insights into conditions found in the general population that continue to remain incompletely understood.

Anti-Lipid IgG Antibodies Are Produced via Germinal Centers in a Murine Model Resembling Human Lupus.

Anti-lipid IgG antibodies are produced in some mycobacterial infections and in certain autoimmune diseases [such as anti-phospholipid syndrome, systemic lupus erythematosus (SLE)]. However, few studies have addressed the B cell responses underlying the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a murine model resembling human lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membranes of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which appear to be involved in the development of the mouse model of lupus. Of note, anti-NPA antibodies are also detected in patients with SLE and leprosy. We used this model of lupus to investigate in vivo the cellular mechanisms that lead to the production of anti-lipid, class-switched IgG antibodies. In this murine lupus model, we found plasma cells (Gr1−, CD19−, CD138+) producing NPA-specific IgGs in the draining lymph nodes, the spleen, and the bone marrow. We also found a significant number of germinal center B cells (IgD−, CD19+, PNA+) specific for NPA in the draining lymph nodes and the spleen, and we identified in situ the presence of NPA in these germinal centers. By contrast, very few NPA-specific, extrafollicular reaction B cells (B220+, Blimp1+) were found. Moreover, when assessing the anti-NPA IgG antibodies produced during the experimental protocol, we found that the affinity of these antibodies progressively increased over time. Altogether, our data indicate that, in this murine model resembling human lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers.

B cells respond via germinal centers to produce anti-lipid IgG antibodies in a murine model of lupus

Abstract Anti-lipid IgG antibodies are produced in some mycobacterial infections and autoimmune diseases (lupus, anti-phospholipid syndrome). However, few studies have addressed the mechanisms that lead to the production of these immunoglobulins. Anti-lipid IgG antibodies are consistently found in a mouse model of lupus induced by chlorpromazine-stabilized non-bilayer phospholipid arrangements (NPA). NPA are transitory lipid associations found in the membrane of most cells; when NPA are stabilized they can become immunogenic and induce specific IgG antibodies, which are involved in the development of the disease. We used this model of lupus to investigate the in vivo mechanisms that lead to the production of anti-lipid IgG antibodies. B cells are activated, in response to most protein antigens, via germinal centers or extrafollicular reactions. In germinal centers, a T cell-dependent response leads to isotype switch, somatic hypermutation, affinity maturation and memory generation, whereas these events do not usually occur in extrafollicular reactions. In this mouse model of lupus induced by chlorpromazine-stabilized NPA, we found plasma cells producing NPA-specific IgGs in the inguinal lymph nodes, the spleen, and bone marrow of mice. We also found a significant number of germinal center B cells specific for NPA in the inguinal lymph nodes and the spleen, and we demonstrated the presence of NPA in these same germinal centers. In contrast, we found very few extrafollicular reaction B cells specific for NPA. Altogether, our data suggest that, in this murine model of lupus, B cells produce anti-NPA IgG antibodies mainly via germinal centers.

A Liposome-Based Approach to the Integrated Multi-Component Antigen Microarrays.

This report describes an experimental procedure for constructing integrated lipid, carbohydrate, and protein microarrays. In essence, it prints liposomes on nitrocellulose-coated micro-glass slides, a biochip substrate for spotting protein and carbohydrate microarrays, and the substances that can form liposomes (homo-liposomes) or can be incorporated into liposomes (hetero-liposomes) are suitable for microarray construction using existing microarray spotting devices. Importantly, this technology allows simultaneous detection of serum antibody activities among the three major classes of antigens, i.e., lipids, carbohydrates, and proteins. The potential of this technology is illustrated by its use in revealing a broad-spectrum of pre-existing anti-lipid antibodies in blood circulation and monitoring the epitope spreading of autoantibody reactivities among protein, carbohydrate, and lipid antigens in experimental autoimmune encephalomyelitis (EAE).

Rapid Treponema pallidum clearance from blood and ulcer samples following single dose benzathine penicillin treatment of early syphilis.

Currently, the efficacy of syphilis treatment is measured with anti-lipid antibody tests. These can take months to indicate cure and, as a result, syphilis treatment trials require long periods of follow-up. The causative organism, Treponema pallidum (T. pallidum), is detectable in the infectious lesions of early syphilis using DNA amplification. Bacteraemia can likewise be identified, typically in more active disease. We hypothesise that bacterial clearance from blood and ulcers will predict early the standard serology-measured treatment response and have developed a qPCR assay that could monitor this clearance directly in patients with infectious syphilis. Patients with early syphilis were given an intramuscular dose of benzathine penicillin. To investigate the appropriate sampling timeframe samples of blood and ulcer exudate were collected intensively for T. pallidum DNA (tpp047 gene) and RNA (16S rRNA) quantification. Sampling ended when two consecutive PCRs were negative. Four males were recruited. The mean peak level of T. pallidum DNA was 1626 copies/ml whole blood and the mean clearance half-life was 5.7 hours (std. dev. 0.53). The mean peak of 16S rRNA was 8879 copies/ml whole blood with a clearance half-life of 3.9 hours (std. dev. 0.84). From an ulcer, pre-treatment, 67,400 T. pallidum DNA copies and 7.08x107 16S rRNA copies were detected per absorbance strip and the clearance half-lives were 3.2 and 4.1 hours, respectively. Overall, T. pallidum nucleic acids were not detected in any sample collected more than 56 hours (range 20–56) after treatment. All patients achieved serologic cure. In patients with active early syphilis, measuring T. pallidum levels in blood and ulcer exudate may be a useful measure of treatment success in therapeutic trials. These laboratory findings need confirmation on a larger scale and in patients receiving different therapies.

Type II NKT-TFH cells against Gaucher lipids regulate B-cell immunity and inflammation

AbstractChronic inflammation including B-cell activation is commonly observed in both inherited (Gaucher disease [GD]) and acquired disorders of lipid metabolism. However, the cellular mechanisms underlying B-cell activation in these settings remain to be elucidated. Here, we report that β-glucosylceramide 22:0 (βGL1-22) and glucosylsphingosine (LGL1), 2 major sphingolipids accumulated in GD, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT) cells. Human βGL1-22– and LGL1-reactive CD1d tetramer–positive T cells have a distinct T-cell receptor usage and genomic and cytokine profiles compared with the classical type I NKT cells. In contrast to type I NKT cells, βGL1-22– and LGL1-specific NKT cells constitutively express T-follicular helper (TFH) phenotype. Injection of these lipids leads to an increase in respective lipid-specific type II NKT cells in vivo and downstream induction of germinal center B cells, hypergammaglobulinemia, and production of antilipid antibodies. Human βGL1-22– and LGL1-specific NKT cells can provide efficient cognate help to B cells in vitro. Frequency of LGL1-specific T cells in GD mouse models and patients correlates with disease activity and therapeutic response. Our studies identify a novel type II NKT-mediated pathway for glucosphingolipid-mediated dysregulation of humoral immunity and increased risk of B-cell malignancy observed in metabolic lipid disorders.

Type II NKT-TFH Cells Against Gaucher Lipids Regulate B Cell Immunity and Inflammation

Gaucher disease (GD) is a lysosomal storage disorder due to an inherited deficiency of acid β-glucosidase (GBA) that leads to the accumulation of two key sphingolipids, glucosylceramide (βGL1) and glucosylsphingosine (LGL1) in lysosomes of mononuclear phagocytes. Pathophysiology of classic GD is complex and includes chronic inflammation with an increased risk of B cell malignancies, pointing to immune dysregulation beyond the accumulation of lipid-loaded macrophages. Although an involvement of accumulated lipids has been implicated, the mechanisms linking sphingolipid accumulation with the observed clinical phenotypes are poorly understood.Natural Killer T (NKT) cells are a specialized lineage of T cells that recognize lipid/glycolipid antigens presented by MHC-like molecule CD1d. NKT cells are currently classified into two major subsets- type I or invariant NKT (iNKT) cells that express a conserved TCR and recognize α-galactosylceramide (α-GalCer) and type II or diverse NKT (dNKT) cells that utilize diverse TCR and do not recognize α-GalCer. Type II NKT cells comprise a major subset in humans and are increasingly implicated in immune regulation of diverse disease states. However in contrast to type I NKT cells, information on the functional properties of human type II NKT cells and their dysregulation in the context of disease is extremely limited.We utilized human and murine CD1d-tetramers loaded with GD-associated lipids (βGL1 and LGL1) to characterize the genomic and functional properties of T cells against these lipids. Analysis of human PBMCs revealed that βGL1-22/LGL1-tetramer+ cells are a distinct subset of CD1d-restricted T cells with a naïve phenotype and diverse Vβ receptor usage. Gene expression profiling as well as multiplex cytokine analysis demonstrated a distinct genomic and cytokine profile of these cells compared to classical type I NKT cells. Using both CD1d blocking experiments and CD1d-expressing C1R cells we could show that βGL1-22 and LGL1 could activate respective lipid-specific T cells in a CD1d dependent manner. Akin to human PBMCs, murine βGL1-22/LGL1 was shown to be a cognate antigen of type II NKT cells by performing double staining with α-GalCer and βGL1-22/LGL1-loaded-CD1d tetramers and using CD1d-/- and Jα18-/- mice. Transcriptional profile of βGL1-22 and LGL1-specific T cells revealed increased expression of genes associated with T-follicular-helper (TFH) phenotype. Therefore, we analyzed whether βGL1-22 or LGL1-specific type II NKT cells acquire TFH phenotype (CXCR5hi PD1hi ICOShi BCL6+IL-21+) upon immunization with βGL1-22 or LGL1. Separate groups of WT (C57BL/6), CD1d-/- and Jα18-/- mice were immunized with vehicle, α-GalCer, βGL1-22 or LGL1, after 7 days splenocytes and serum from each mice strain was assessed for upregulation of TFH markers and germinal center (GC) B cells by flow cytometry and antibodies by ELISA respectively. In contrast to type-I NKT cells, βGL1-22 and LGL1-specific NKT cells constitutively expressed TFH phenotype. The magnitude of TFH response was dictated by the amount of antigen and directly correlated with induction of GC B cells, hypergammaglobulinemia and production of anti-lipid antibodies. Complementarily, in vitro co culture of human βGL1-22 and LGL1-specific type II NKT cells with purified autologous B cells induced TFH markers in lipid specific T cells and led to expansion of plasmablasts and Ig secretion. In order to study changes in βGL1-22/LGL1-specific T cells in the setting of a disease wherein these ligands are altered, we analyzed patients and mouse models with GD. Compared to wild type control mice, GD mice exhibited >20-fold increase of LGL1-specific T cells. Concomitant to increase in LGL1-specific T cells in GD mice, notable up regulation of TFH markers and induction of anti-lipid antibodies was also observed. Similar to GD mice, sphingolipids accumulation in GD patients results in an increase of LGL1-specific type II NKT cells which correlated with biomarkers of disease severity and show reduction upon clinical improvement with enzyme replacement therapy.These data reveal and characterize a new subset of human and murine type II NKT-TFH cells against lipids dysregulated in Gaucher disease that regulate B cell immunity and inflammation and provide a mechanistic link between lipid storage and inflammation. DisclosuresPastores:Genzyme: Honoraria, Research Funding; Shire: Honoraria, Research Funding. Mistry:Genzyme, a Sanofi Company: Consultancy, Honoraria, Travel reimbursement, Research grants Other. Dhodapkar:Celgene: Research Funding.

Correction: Lipid Anti-Lipid Antibody Responses Correlate with Disease Activity in Systemic Lupus Erythematosus

Correction: Lipid Anti-Lipid Antibody Responses Correlate with Disease Activity in Systemic Lupus Erythematosus

Antiphospholipid IgM antibody response in acute and chronic Mycobacterium tuberculosis mouse infection model

The clinical management of tuberculosis (TB) could be greatly improved by an affordable biomarker test to monitor treatment response. Here, we examined changes in immunoglobulin M (IgM) antibody response to lipids as a potential biomarker for monitoring TB treatment in an experimental mouse model. We performed enzyme-linked immunosorbent assay to investigate changes in IgM antibody response against cardiolipin (CL), phosphatidylcholine (PTC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and sphingolipid (SL) in BALB/c mice that were treated after being infected with Mycobacterium tuberculosis for 4 weeks (acute infection) and 20 weeks (chronic infection). Cytokine levels [interleukin (IL)-5, IL-10, interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1)] in lung and spleen homogenates as well as in blood were also compared. In both acutely and chronically infected mice, lungs were sterilised of M. tuberculosis infection after 8 weeks of treatment. The IgM response to CL, PTC, PE, PI and SL were consistently elevated throughout the course of infection in chronically infected mice compared with acutely infected mice. In acutely infected mice, the IgM antibody response against CL significantly decreased after 8 weeks of treatment, but not against other lipids. In chronically infected mice, the IgM response showed no significant changes against any of the lipids after 8 weeks of treatment. Of the cytokines examined, only MCP-1 levels in lungs decreased significantly after treatment. These findings demonstrate that antilipid IgM antibody can remain elevated in chronically infected mice, but with treatment, only anti-CL IgM antibody levels decreased together with M. tuberculosis bacterial burden in acutely infected mice. Treatment did not affect antilipid IgM levels in chronically infected mice.

Novel phage display-derived mycolic acid-specific antibodies with potential for tuberculosis diagnosis

Tuberculosis is a major cause of mortality and morbidity due to infectious disease. However, current clinical diagnostic methodologies such as PCR, sputum culture, or smear microscopy are not ideal. Antibody-based assays are a suitable alternative but require specific antibodies against a suitable biomarker. Mycolic acid, which has been found in patient sputum samples and comprises a large portion of the mycobacterial cell wall, is an ideal target. However, generating anti-lipid antibodies using traditional hybridoma methodologies is challenging and has limited the exploitation of this lipid as a diagnostic marker. We describe here the isolation and characterization of four anti-mycolic acid antibodies from a nonimmune antibody phage display library that can detect mycolic acids down to a limit of 4.5ng. All antibodies were specific for the methoxy subclass of mycolic acid with weak binding for α mycolic acid and did not show any binding to closely related lipids or other Mycobacterium tuberculosis (Mtb) derived lipids. We also determined the clinical utility of these antibodies based on their limit of detection for mycobacteria colony forming units (CFU). In combination with an optimized alkaline hydrolysis method for rapid lipid extraction, these antibodies can detect 10(5) CFU of Mycobacterium bovis BCG, a close relative of Mtb and therefore represent a novel approach for the development of diagnostic assays for lipid biomarkers.

O05.2 Measuring Syphilis: Quantitative PCR Can Be Used to Monitor Treatment Response

BackgroundThe humoral response to Treponema pallidum ( T. pallidum), which causes syphilis, is divided into ‘non-specific’ anti-lipid and specific anti-treponemal protein antibodies. A four-fold reduction in anti-lipid antibodies is used...

Lateral diffusion of peripheral membrane proteins on supported lipid bilayers is controlled by the additive frictional drags of (1) bound lipids and (2) protein domains penetrating into the bilayer hydrocarbon core

Peripheral membrane proteins bound to lipids on bilayer surfaces play central roles in a wide array of cellular processes, including many signaling pathways. These proteins diffuse in the plane of the bilayer and often undergo complex reactions involving the binding of regulatory and substrate lipids and proteins they encounter during their 2D diffusion. Some peripheral proteins, for example pleckstrin homology (PH) domains, dock to the bilayer in a relatively shallow position with little penetration into the bilayer. Other peripheral proteins exhibit more complex bilayer contacts, for example classical protein kinase C isoforms (PKCs) bind as many as six lipids in stepwise fashion, resulting in the penetration of three PKC domains (C1A, C1B, C2) into the bilayer headgroup and hydrocarbon regions. A molecular understanding of the molecular features that control the diffusion speeds of proteins bound to supported bilayers would enable key molecular information to be extracted from experimental diffusion constants, revealing protein–lipid and protein–bilayer interactions difficult to study by other methods. The present study investigates a range of 11 different peripheral protein constructs comprised by 1–3 distinct domains (PH, C1A, C1B, C2, anti-lipid antibody). By combining these constructs with various combinations of target lipids, the study measures 2D diffusion constants on supported bilayers for 17 different protein–lipid complexes. The resulting experimental diffusion constants, together with the known membrane interaction parameters of each complex, are used to analyze the molecular features correlated with diffusional slowing and bilayer friction. The findings show that both (1) individual bound lipids and (2) individual protein domains that penetrate into the hydrocarbon core make additive contributions to the friction against the bilayer, thereby defining the 2D diffusion constant. An empirical formula is developed that accurately estimates the diffusion constant and bilayer friction of a peripheral protein in terms of its number of bound lipids and its geometry of penetration into the bilayer hydrocarbon core, yielding an excellent global best fit (R2 of 0.97) to the experimental diffusion constants. Finally, the observed additivity of the frictional contributions suggests that further development of current theory describing bilayer dynamics may be needed. The present findings provide constraints that will be useful in such theory development.