LEGACY: Phase 2a Trial to Evaluate the Safety, Pharmacokinetics, and Pharmacodynamic Effects of the Anti-EL (Endothelial Lipase) Antibody MEDI5884 in Patients With Stable Coronary Artery Disease.

OBJECTIVE: Functional HDL (high-density lipoprotein) particles that facilitate cholesterol efflux may be cardioprotective. EL (endothelial lipase) hydrolyzes phospholipids promoting catabolism of HDL and subsequent renal excretion. MEDI5884 is a selective, humanized, monoclonal, EL-neutralizing antibody. We sought to determine the safety, pharmacokinetics, and pharmacodynamic effects of multiple doses of MEDI5884 in patients with stable coronary artery disease. Approach and Results: LEGACY was a phase 2a, double-blind, placebo-controlled, parallel-design trial that randomized 132 patients with stable coronary artery disease receiving high-intensity statin therapy to 3 monthly doses of 1 of 5 dose levels of MEDI5884 (50, 100, 200, 350, or 500 mg SC) or matching placebo. The primary end point was the safety and tolerability of MEDI5884 through the end of the study (day 151). Additional end points included change in HDL cholesterol and cholesterol efflux from baseline to day 91, hepatic uptake of cholesterol at day 91, changes in various other lipid parameters. The incidence of adverse events was similar between the placebo and MEDI5884 groups. In a dose-dependent manner, MEDI5884 increased HDL cholesterol up to 51.4% (P<0.0001) and global cholesterol efflux up to 26.2% ([95% CI, 14.3-38.0] P<0.0001). MEDI5884 increased HDL particle number up to 14.4%. At the highest dose tested, an increase in LDL (low-density lipoprotein) cholesterol up to 28.7% (P<0.0001) and apoB (apolipoprotein B) up to 13.1% (P=0.04) was observed with MEDI5884. However, at the potential target doses for future studies, there was no meaningful increase in LDL cholesterol or apoB. CONCLUSIONS: Inhibition of EL by MEDI5884 increases the quantity and quality of functional HDL in patients with stable coronary artery disease on high-intensity statin therapy without an adverse safety signal at the likely dose to be used. These data support further clinical investigation. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03351738.

A natural symbiotic bacterium drives mosquito refractoriness to Plasmodium infection via secretion of an antimalarial lipase.

The stalling global progress in the fight against malaria prompts the urgent need to develop new intervention strategies. Whilst engineered symbiotic bacteria have been shown to confer mosquito resistance to parasite infection, a major challenge for field implementation is to address regulatory concerns. Here, we report the identification of a Plasmodium-blocking symbiotic bacterium, Serratia ureilytica Su_YN1, isolated from the midgut of wild Anopheles sinensis in China that inhibits malaria parasites via secretion of an antimalarial lipase. Analysis of Plasmodium vivax epidemic data indicates that local malaria cases in Tengchong (Yunnan province, China) are significantly lower than imported cases and importantly, that the local vector A. sinensis is more resistant to infection by P. vivax than A. sinensis from other regions. Analysis of the gut symbiotic bacteria of mosquitoes from Yunnan province led to the identification of S. ureilytica Su_YN1. This bacterium renders mosquitoes resistant to infection by the human parasite Plasmodium falciparum or the rodent parasite Plasmodium berghei via secretion of a lipase that selectively kills parasites at various stages. Importantly, Su_YN1 rapidly disseminates through mosquito populations by vertical and horizontal transmission, providing a potential tool for blocking malaria transmission in the field.

Decreased T-cell mediated hepatic injury in concanavalin A-treated PLRP2-deficient mice.

Concanavalin A (Con A) activates innate immunity and causes liver damage mediated by cytotoxic T lymphocytes (CTL) in mice. The Pancreatic lipase-related protein 2 (PLRP2) is induced by interleukin (IL)-4 in vitro in CTLs and associated with CTL functions. We examined the role of PLRP2 in a mouse model of Con A-induced T cell-mediated hepatitis. PLRP2-knockout and wild-type (WT) mice were inoculated with 20 mg/kg Con A. Mice lacking PLRP2 reduced Con A-induced hepatitis, which was manifested by a decrease in serum aminotransferase and histopathological assessment. The expression and secretion of cytokines including tumor necrosis factor-alpha (TNF-α), interferon (IFN)-γ, IL-6, and IL-1ß were suppressed in Con A-treated PLRP2-knockout mice. In PLRP2 knockout mice, Con A-induced liver chemokines and adhesion molecules (such as MIP-1α, MIP-1ß, ICAM-1 and MCP-1) were also down regulated. In the WT liver treated with Con A, the number of T cells (CD4+ and CD8+) and macrophages (CD11b+ F4/80+) increased significantly, while the lack of PLRP2 reduced the number of T cells in the liver, but had no effect on macrophages. The shift of the metabolic profiles was impaired in Con A-treated PLRP2-knockout mice compared to WT mice. In conclusion, these results indicate that PLRP2 deficiency reduces T-cell mediated Con A-induced hepatitis, and suggest PLRP2 is a potential target of anti-inflammatory and immunomodulatory drugs to treat immune-mediated hepatitis.

Histopathological findings and canine pancreatic lipase immunoreactivity in normal dogs and dogs with inflammatory and neoplastic diseases of the pancreas.

BACKGROUND: Diagnosis of pancreatic diseases in dogs is still challenging because of variable clinical signs, which do not always correspond with clinical pathology and histopathological findings. OBJECTIVES: To characterize inflammatory and neoplastic pancreatic diseases of dogs and to correlate these findings with clinical findings and canine pancreatic lipase immunoreactivity (cPLI) results. ANIMALS: Tissue specimens and corresponding blood samples from 72 dogs submitted for routine diagnostic testing. METHODS: Four groups were defined histologically: (1) normal pancreas (n = 40), (2) mild pancreatitis (n = 8), (3) moderate or severe pancreatitis (acute, n = 11; chronic, n = 1), and (4) pancreatic neoplasms (n = 12). An in-house cPLI ELISA (<180 µg/L, normal; >310 µg/L, pancreatitis) was performed. RESULTS: In dogs with normal pancreas, 92.5% of serum cPLI results were within the reference range and significantly lower than in dogs with mild acute pancreatitis, moderate or severe acute pancreatitis and pancreatic tumors. In dogs with moderate or severe acute pancreatitis, cPLI sensitivity was 90.9% (95% confidence interval [CI], 58.7%-99.8%). Most dogs (9/12) with pancreatic tumors (group 4) had additional pancreatic inflammation and cPLI results were increased in 10 dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: High cPLI indicates serious acute pancreatitis but underlying pancreatic neoplasms should also be taken into consideration. This study confirms the relevance of histopathology in the diagnostic evaluation of pancreatic diseases.

Comparative repeatability of pancreatic lipase assays in the commercial and in-house laboratory environments.

BACKGROUND: Sensitivity and specificity for commercial and in-house pancreatic lipase immunoreactivity (cPLI) assays have been reported, but repeatability under routine clinical conditions is unknown. OBJECTIVES: To determine: Coefficient of variation (CV) between replicates of a commercial assay (Spec cPL) and 2 in-house assays (VetScan cPL, Vcheck cPL) under routine conditions. Effects of sample condition or personnel on results. Potential directional bias between assays. ANIMALS: Serum from 12 canine clinical patients. METHODS: Prospective study. Serum Spec cPL, VetScan cPL, and Vcheck cPL (6 aliquots each) were measured, and CVs were calculated, effects of sample condition and personnel were assessed using a linear mixed model, and direction of bias was assessed using least square mean cPLI concentration. RESULTS: Mean %CVs for Spec cPL, VetScan cPL, and Vcheck cPL were 5.5, 17.0, and 23.7%. Three of 6 VetScan cPL samples and 5/9 Vcheck cPL samples had an unacceptably high %CV (>20%). Transportation (Spec cPL) and sample condition or personnel (VetScan cPL, Vcheck cPL) did not affect repeatability. Least square mean cPL was higher for Spec cPL (807.9 µg/L) than for VetScan cPL (558.5 µg/L) or Vcheck cPL (399.8 µg/L). CONCLUSIONS AND CLINICAL IMPORTANCE: For clinical use, the commercial Spec cPL has the highest repeatability, and Vcheck cPL has significantly lower repeatability. Both in-house assays evaluated may provide discrepant categorical results ("pancreatitis" versus "equivocal" versus "not pancreatitis") for the same sample. In-house pancreatic lipase concentrations may be lower than those determined by the Spec cPL assay.

A twist of FATe: Lipid droplets and inflammatory lipid mediators.

Lipid droplets are fat storage organelles present in most eukaryotic cells. They consist of a neutral lipid core containing mostly triglycerides and sterol esters and covered by a monolayer of phospholipids, wherein numerous proteins are embedded. In the cell, lipid droplets have a dynamic life cycle, rapidly altering their size, location, lipid and protein composition in response to environmental stimuli and cell state. Lipid droplets are primarily involved in the coordination of lipid metabolism with cellular requirements for energy production, membrane homeostasis and cell growth. However, they are also directly or indirectly engaged in signalling pathways. On the one hand, lipid droplets sequester lipids and proteins thereby limiting their availability for participation in signalling pathways. On the other hand, the lipolytic machinery provides a highly regulated, on-demand source of signalling lipids: lipids derived from their neutral lipid core, or the phospholipid monolayer, directly act as signalling mediators or are converted into ones. In fact, emerging studies suggest that these organelles are essential for various cellular stress response mechanisms, including inflammation and immunity, acting as hubs that integrate metabolic and inflammatory processes. Here, we discuss the ways in which lipid droplets regulate the availability of fatty acids for the activation of signalling pathways and for the production of polyunsaturated fatty acid-derived lipid mediators. We focus in particular on recent discoveries in immune cells and adipose tissue that have revealed an intricate relationship between lipid droplets and inflammatory signalling and may also be relevant for other tissues and various human diseases.

Bacterial lipolysis of immune-activating ligands promotes evasion of innate defenses.

Commensal and pathogenic bacteria hydrolyze host lipid substrates with secreted lipases and phospholipases for nutrient acquisition, colonization, and infection. Bacterial lipase activity on mammalian lipids and phospholipids can promote release of free fatty acids from lipid stores, detoxify antimicrobial lipids, and facilitate membrane dissolution. The gram-positive bacterium Staphylococcus aureus secretes at least two lipases, Sal1 and glycerol ester hydrolase (Geh), with specificities for short- and long-chain fatty acids, respectively, each with roles in the hydrolysis of environmental lipids. In a recent study from our group, we made the unexpected observation that Geh released by S. aureus inhibits activation of innate immune cells. Herein, we investigated the possibility that S. aureus lipases interface with the host immune system to blunt innate immune recognition of the microbe. We found that the Geh lipase, but not other S. aureus lipases, prevents activation of innate cells in culture. Mutation of geh leads to enhancement of proinflammatory cytokine production during infection, increased innate immune activity, and improved clearance of the bacterium in infected tissue. These in vitro and in vivo effects on innate immunity were not due to direct functions of the lipase on mammalian cells, but rather a result of inactivation of S. aureus lipoproteins, a major pathogen-associated molecular pattern (PAMP) of extracellular gram-positive bacteria, via ester hydrolysis. Altogether, these studies provide insight into an adaptive trait that masks microbial recognition by innate immune cells through targeted inactivation of a broadly conserved PAMP.

Effects of immunosuppressive prednisolone therapy on pancreatic tissue and concentration of canine pancreatic lipase immunoreactivity in healthy dogs.

The objective of this study was to examine the effects of immunosuppressive prednisolone therapy on pancreatic tissue and the concentration of serum canine pancreatic lipase immunoreactivity (cPLI) in healthy dogs. Six healthy beagle dogs were subcutaneously administered an immunosuppressive dose of prednisolone [4 mg/kg body weight (BW)] once daily for either 2 or 3 weeks. Serum cPLI concentration was measured before and after treatment. Ultrasonographic examination of the pancreas and laparoscopic biopsy and histopathological examination of the right pancreatic lobe and the liver were also conducted before and after treatment. The expression of pancreatic lipase messenger ribonucleic acid (mRNA) in the pancreas and liver was examined by polymerase chain reaction (PCR). Although the serum cPLI concentration was significantly higher on day 14 and on the day of the second laparoscopy than before treatment, it was classified as normal (≤ 200 µg/L) in 5 dogs and as abnormal (≥ 400 µg/L) in only 1 dog. None of the 6 dogs showed clinical signs of pancreatitis during the study period. After treatment, ultrasonographic examination of the pancreas showed no changes except for a hypoechoic pancreas in 1 dog. Histopathological examination of the right pancreatic lobe in all dogs showed no evidence of pancreatitis after treatment. Pancreatic lipase mRNA expression was detected in the pancreas, but not in the liver, before and after treatment. The administration of 4 mg/kg BW per day of prednisolone for 2 or 3 weeks increased the serum cPLI concentration without clinical signs of pancreatitis, although an abnormal cPLI concentration (≥ 400 µg/L) was observed in only 1 dog. No ultrasonographic or histological evidence of pancreatitis was observed in any of the dogs.

L'objectif de la présente étude était d'examiner les effets d'une thérapie immunosuppressive par la prednisolone sur le tissu pancréatique et la concentration sérique canine de lipase pancréatique immunoréactive (cPLI) chez des chiens en santé. Six chiens beagle en santé ont reçu par voie sous-cutanée une dose immunosuppressive de prednisolone [4 mg/kg de poids corporel (PC)] une fois par jour pendant 2 ou 3 semaines. La concentration sérique de cPLI a été mesurée avant et après le traitement. Un examen échographique du pancréas et une biopsie suivie d'un examen histopathologique d'échantillons du lobe pancréatique droit ainsi que du foie obtenus par laparoscopie ont également été faits avant et après le traitement. L'expression de l'ARNm de la lipase pancréatique dans le pancréas et le foie a été examinée par réaction d'amplification en chaine par la polymérase. Bien que la concentration sérique de cPLI fût significativement plus élevée au jour 14 et le jour de la seconde laparoscopie qu'avant le traitement, elle était classée comme normale (≤ 200 µg/L) chez cinq chiens et comme anormale (≥ 400 µg/L) chez seulement un chien. Aucun des six chiens n'a présenté de signes cliniques de pancréatite durant la période d'étude. Après le traitement, l'examen échographique du pancréas ne démontrait aucun changement sauf pour un pancréas hypoéchogène chez un chien. L'examen histopathologique du lobe pancréatique droit chez tous les chiens n'a pas permis de mettre en évidence de pancréatite après le traitement. L'expression d'ARNm de lipase pancréatique fut détectée dans le pancréas, mais pas dans le foie, avant et après le traitement. L'administration de 4 mg/kg de PC par jour de prednisolone pendant 2 ou 3 semaines a fait augmenter la concentration sérique de cPLI sans signe clinique de pancréatite, bien qu'une concentration anormale de cPLI (≥ 400 µg/L) fût obtenue chez un chien. Aucune évidence échographique ou histologique de pancréatite ne fût observée chez les chiens de cette étude.(Traduit par Docteur Serge Messier).

Reduction in White Blood Cell, Neutrophil, and Red Blood Cell Counts Related to Sex, HLA, and Islet Autoantibodies in Swedish TEDDY Children at Increased Risk for Type 1 Diabetes.

Islet autoantibodies (IAs) precede the clinical onset of type 1 diabetes (T1D); however, the knowledge is limited about whether the prodrome affects complete blood counts (CBCs) in 4- to 12-year-old children with increased genetic risk for T1D. This study tested whether CBCs were altered in 4- to 12-year-old children without (n = 376) or with one or several IAs against insulin, GAD65, or IA-2 (n = 72). CBC was analyzed during longitudinal follow-up in 448 Swedish children enrolled in The Environmental Determinants of Diabetes in the Young (TEDDY) study. A linear mixed-effects model was used to assess potential association between IA and CBC measurements over time. The white blood cell and neutrophil counts were reduced in children with IAs, primarily in boys. In contrast, girls had lower levels of hemoglobin and hematocrit. Positivity for multiple IAs showed the lowest counts in white blood cells and neutrophils in boys and red blood cells, hemoglobin, and hematocrit in girls. These associations were primarily observed in children with the HLA-DR3-DQ2/DR4-DQ8 genotype. We conclude that the reduction in neutrophils and red blood cells in children with multiple IAs and HLA-DR3-DQ2/DR4-DQ8 genotype may signal a sex-dependent islet autoimmunity detected in longitudinal CBCs.

LPS inactivation by a host lipase allows lung epithelial cell sensitization for allergic asthma.

Allergic asthma is a chronic inflammatory disease primarily mediated by Th2 immune mechanisms. Numerous studies have suggested that early life exposure to lipopolysaccharide (LPS) is negatively associated with allergic asthma. One proposed mechanism invokes desensitization of lung epithelial cells by LPS. We report here that acyloxyacyl hydrolase (AOAH), a host lipase that degrades and inactivates LPS, renders mice more susceptible to house dust mite (HDM)-induced allergic asthma. Lung epithelial cells from Aoah-/- mice are refractory to HDM stimulation, decreasing dendritic cell activation and Th2 responses. Antibiotic treatment that diminished commensal LPS-producing bacteria normalized Aoah-/- responses to HDM, while giving LPS intrarectally ameliorated asthma. Aoah-/- mouse feces, plasma, and lungs contained more bioactive LPS than did those of Aoah+/+ mice. By inactivating commensal LPS, AOAH thus prevents desensitization of lung epithelial cells. An enzyme that prevents severe lung inflammation/injury in Gram-negative bacterial pneumonia has the seemingly paradoxical effect of predisposing to a Th2-mediated airway disease.

Canine pancreatic lipase immunoreactivity concentrations associated with intervertebral disc disease in 84 dogs.

OBJECTIVE: To determine the differences in serum canine pancreatic lipase immunoreactivity between dogs with intervertebral disc herniation and healthy control dogs. MATERIALS AND METHODS: Eighty-four client-owned dogs with intervertebral disc herniation, diagnosed by neurologic examination and imaging, and 18 healthy control dogs. Samples of whole blood were collected within 90 minutes of admission. Serum canine pancreatic lipase immunoreactivity concentrations were measured by a commercial immunoassay and evaluated for association with intervertebral disc herniation, signalment, neurolocalisation and the preadmission administration of glucocorticosteriods or non-steroidal anti-inflammatory drugs. RESULTS: Serum canine pancreatic lipase immunoreactivity concentrations were statistically increased in dogs with intervertebral disc herniation (P<0·01, n=38). A subgroup of dogs (19/38) with elevated canine pancreatic lipase immunoreactivity concentrations was re-evaluated between 2 and 4 weeks later, and 15 had resolution of clinical signs and values less than 200 µg/L. Serum canine pancreatic lipase immunoreactivity concentrations were not significantly correlated with clinical gastrointestinal disease, neurolocalisation or the preadmission administration of corticosteroids or non-steroidal anti-inflammatory drugs. CLINICAL SIGNIFICANCE: These results suggest that serum canine pancreatic lipase immunoreactivity concentrations are significantly elevated in dogs with intervertebral disc herniation.

Newly developed selective immunoinactivation assay revealed reduction in adipose triglyceride lipase activity in peripheral leucocytes from patients with idiopathic triglyceride deposit cardiomyovasculopathy.

Triglyceride deposit cardiomyovasculopathy (TGCV) is a rare and newly identified disease among patients requiring cardiac transplantation. TGCV is characterized by cardiomyocyte steatosis and triglyceride (TG)-deposit atherosclerosis, resulting from the abnormal intracellular metabolism of TG. TGCV is classified into primary and idiopathic types. Primary TGCV carries ultra-rare genetic mutations in the adipose triglyceride lipase (ATGL), a rate-liming enzyme that hydrolyzes intracellular TG in adipose and non-adipose tissues. Idiopathic TGCV, first identified among autopsied individuals with diabetes mellitus (DM) with severe heart diseases, shows no ATGL mutations and its causes and underlying mechanisms are still unknown. TGCV is difficult to diagnose in daily clinics, thereby demanding feasible diagnostic procedures. We aimed to develop an assay to measure ATGL activity using peripheral leucocytes. Human his6-ATGL was expressed in COS1 cells, purified to homogeneity, and used to raise a polyclonal antibody neutralizing TG-hydrolyzing activity of ATGL. We developed a selective immunoinactivation assay (SIIA) for the quantitation of ATGL activity in cell lysates of leucocytes by the antibody neutralizing ATGL activities. ATGL activity was measured in 13 idiopathic TGCV patients, with two patients with primary TGCV as the negative control. Healthy (non-DM) and DM controls without heart diseases were also subjected. The developed SIIA assay revealed significant reduction in ATGL activity in leucocytes from patients with idiopathic TGCV who did not carry ATGL mutations as compared with non-DM and DM controls. Thus, ATGL in leucocytes may be an important biomarker for the diagnosis of TGCV and our assay may provide insights into pathophysiology and elucidate the underlying mechanism of TGCV and related disorders.

A new enzyme-linked immunosorbent assay system for human serum hepatic triglyceride lipase.

There is no established method for measuring human hepatic triglyceride (TG) lipase (HTGL) concentration in serum. In this study, we developed new monoclonal Abs (MoAbs) (9A1 mouse MoAb and 141A1 rat MoAb) that react with HTGL both in serum and in postheparin plasma (PHP) and established a novel ELISA system for measuring serum HTGL and PHP-HTGL concentrations. To confirm the specificity of MoAbs, we performed immunoprecipitation-immunoblotting analysis. Both 9A1 mouse MoAb and 141A1 rat MoAb were able to immunoprecipitate not only recombinant HTGL and PHP-HTGL but also serum HTGL, demonstrating that HTGL exists in serum obtained without heparin injection. This method yielded intra- and interassay coefficients of variation of <6% and showed no cross-reactivity with LPL or endothelial lipase. In clinical analysis on 42 male subjects with coronary artery disease, there were strong positive correlations of serum HTGL concentration to PHP-HTGL concentration (r = 0.727, P < 0.01). Serum HTGL concentrations showed positive correlations to serum TGs (r = 0.314, P < 0.05) and alanine aminotransferase (r = 0.406, P < 0.01), and tendencies toward positive correlations to LDL cholesterol, small dense LDL, and γGTP. These results suggest that this new ELISA method for measuring serum HTGL is applicable in daily clinical practice.

Serial measurement of pancreatic lipase immunoreactivity concentration in dogs with immune-mediated disease treated with prednisolone.

OBJECTIVES: In this pilot study, serum canine pancreatic lipase immunoreactivity was measured repeatedly in dogs with various immune-mediated diseases that were treated with immunosuppressive doses of prednisolone. METHODS: Ten client-owned dogs with newly diagnosed immune-mediated disease that had normal canine pancreatic lipase immunoreactivity concentrations (≤200 µg/l) were treated with 2 to 2.2 mg/kg prednisolone orally once daily as the initial treatment. Serum samples were obtained from each of the dogs prior to treatment and at 1- to 4-week intervals during immunosuppressive treatment. The highest canine pancreatic lipase immunoreactivity concentration detected during immunosuppressive treatment was defined as the peak canine pancreatic lipase immunoreactivity. RESULTS: Peak canine pancreatic lipase immunoreactivity concentrations were classified as normal in two dogs, questionable (201 to 399 µg/l) in three dogs, and abnormal (≥400 µg/l) in five dogs. Peak canine pancreatic lipase immunoreactivity concentrations were significantly higher than baseline canine pancreatic lipase immunoreactivity concentrations but there was no evidence of clinical pancreatitis. CLINICAL SIGNIFICANCE: It remains unclear whether the five of 10 dogs with elevated canine pancreatic lipase immunoreactivity during prednisone treatment had subclinical pancreatitis or whether the abnormal results were a consequence of prednisolone administration.

Functional characterization of recombinant major envelope protein (rB2L) of orf virus.

Orf, or contagious ecthyma, a highly contagious transboundary disease of sheep and goats, is caused by a double-stranded DNA virus (ORFV) belonging to the genus Parapoxvirus of the family Poxviridae. The ORFV genome encodes the major envelope proteins B2L and F1L, which have been found to be highly immunogenic and have multiple functional characteristics. In order to investigate the functional properties of the B2L protein, in this study, the B2L gene of ORFV strain 59/05, encoding recombinant mature B2L (aa 1M-D334), was produced as a fusion protein in Escherichia coli. The functional characteristics of purified rB2L fusion protein (~60 kDa) were evaluated in vivo and in vitro, showing that this protein had lipase and immunomodulatory activities. Immunization trials involving laboratory animals (mice, rabbits and guinea pigs) using either constant or graded doses of rB2L fusion protein with or without adjuvants (FCA, alum) as well as co-administration with candidate rErns-Ag protein of classical swine fever virus (CSFV) indicated that the rB2L protein is immunogenic and has immunomodulatory properties. This study shows the potential utility of the rB2L protein as a safe and novel adjuvant in veterinary vaccine formulations.

Identification and Characterization of Lipase Activity and Immunogenicity of LipL from Mycobacterium tuberculosis.

Lipids and lipid-metabolizing esterases/lipases are highly important for the mycobacterial life cycle and, possibly, for mycobacterial virulence. In this study, we expressed 10 members of the Lip family of Mycobacterium tuberculosis. Among the 10 proteins, LipL displayed a significantly high enzymatic activity for the hydrolysis of long-chain lipids. The optimal temperature for the lipase activity of LipL was demonstrated to be 37°C, and the optimal pH was 8.0. The lipase active center was not the conserved motif G-x-S-x-G, but rather the S-x-x-K and GGG motifs, and the key catalytic amino acid residues were identified as G50, S88, and K91, as demonstrated through site-directed mutagenesis experiments. A three-dimensional modeling structure of LipL was constructed, which showed that the GGG motif was located in the surface of a pocket structure. Furthermore, the subcellular localization of LipL was demonstrated to be on the mycobacterial surface by Western blot analysis. Our results revealed that the LipL protein could induce a strong humoral immune response in humans and activate a CD8+ T cell-mediated response in mice. Overall, our study identified and characterized a novel lipase denoted LipL from M. tuberculosis, and demonstrated that LipL functions as an immunogen that activates both humoral and cell-mediated responses.

Serological proteome analysis reveals new specific biases in the IgM and IgG autoantibody repertoires in autoimmune polyendocrine syndrome type 1.

OBJECTIVE: Autoimmune polyendocrine syndrome type 1 (APS 1) is caused by mutations in the AIRE gene that induce intrathymic T-cell tolerance breakdown, which results in tissue-specific autoimmune diseases. DESIGN: To evaluate the effect of a well-defined T-cell repertoire impairment on humoral self-reactive fingerprints, comparative serum self-IgG and self-IgM reactivities were analyzed using both one- and two-dimensional western blotting approaches against a broad spectrum of peripheral tissue antigens. METHODS: Autoantibody patterns of APS 1 patients were compared with those of subjects affected by other autoimmune endocrinopathies (OAE) and healthy controls. RESULTS: Using a Chi-square test, significant changes in the Ab repertoire were found when intergroup patterns were compared. A singular distortion of both serum self-IgG and self-IgM repertoires was noted in APS 1 patients. The molecular characterization of these antigenic targets was conducted using a proteomic approach. In this context, autoantibodies recognized more significantly either tissue-specific antigens, such as pancreatic amylase, pancreatic triacylglycerol lipase and pancreatic regenerating protein 1α, or widely distributed antigens, such as peroxiredoxin-2, heat shock cognate 71-kDa protein and aldose reductase. As expected, a well-defined self-reactive T-cell repertoire impairment, as described in APS 1 patients, affected the tissue-specific self-IgG repertoire. Interestingly, discriminant IgM reactivities targeting both tissue-specific and more widely expressed antigens were also specifically observed in APS 1 patients. Using recombinant targets, we observed that post translational modifications of these specific antigens impacted upon their recognition. CONCLUSIONS: The data suggest that T-cell-dependent but also T-cell-independent mechanisms are involved in the dynamic evolution of autoimmunity in APS 1.

Increased virulence of Mycobacterium tuberculosis H37Rv overexpressing LipY in a murine model.

We have investigated the role of Rv3097c-encoded lipase (LipY) on the virulence of Mycobacterium tuberculosis. It has been shown that the overexpression of LipY in strain H37Rv induced increase in virulence of recombinant H37Rv::LipY strain. Compared to H37Rv, infection with H37Rv::LipY caused enhanced mortality, weight loss, bacterial load in lungs, splenomegaly, worsening lung morphology and pathology. Mice immunized with recombinant LipY antigen were protected against challenge with H37Rv::LipY, which correlated with enhanced survival of challenged mice and striking decrease in pathological features observed in unimmunized mice. To probe the cause of increase in virulence of H37Rv::LipY, the immune status of the host infected with H37Rv and H37Rv::LipY was compared. It was found that overexpression of LipY compromised immune responses resulting in attenuation of Th1 and Th17 responses, significant increase in IL-10, decrease in number of macrophages and T cells, and increase in numbers of Treg, and DCs in the lungs whereas in mice immunized with LipY an increased pool of T cells and DCs was observed. This led us to conclude that the increase in the virulence of H37Rv::LipY was due to downregulation of the host's protective immunity and the Rv3097c encoded LipY lipase is a virulence factor of M. tuberculosis.

Secreted Candida parapsilosis lipase modulates the immune response of primary human macrophages.

Candida parapsilosis is an important opportunistic pathogen with increasing prevalence. Extracellular lipases have been shown to play an important role in the virulence of pathogenic Candida species. However, studying the role of secreted lipase in C. albicans is challenging due to the lack of a mutant strain deficient in all 10 lipase genes. In contrast, we have previously constructed a lipase mutant C. parapsilosis strain lacking both CpLIP1 and CpLIP2, and shown that it has significantly decreased virulence in various infection models, and is killed more efficiently by mouse macrophages. In the present study, we compared the response of human peripheral blood monocyte-derived macrophages to a wild type (wt) as well as a lipase-deficient (lip(-/-)) C. parapsilosis strain that has been previously established in our lab. Although macrophages phagocytosed both strains with similar efficiency, lipase mutants were killed more efficiently according to fluorescent microscopic analysis. The more efficient killing of lip(-/-) cells was confirmed by CFU-determinations. Phagocytosis of wt and lip(-/-)C. parapsilosis was also examined by flow cytometry, revealing that both strains were internelized to the similar extent by macrophages. Additionally, quantitative imaging analysis revealed that the rate of phagolysosome fusion was higher in case of lip(-/-)C. parapsilosis. Interestingly, macrophages stimulated with lip(-/-)C. parapsilosis showed at least 1.5-fold higher expression of TNFα, IL-1ß, IL-6, IL-8, and PTGS-2 after 12 h compared with those infected with wt C. parapsilosis, as determined by qRT-PCR. Furthermore, the lip(-/-)C. parapsilosis strain induced significantly higher TNFα, IL-1ß, IL-6, and IL-10 protein production in macrophages after 24 h compared with the wt strain. These findings confirm the role of fungal lipases as important virulence factors during C. parapsilosis infection.