LIGHT protein: A novel gingival crevicular fluid biomarker associated with increased probing depth after periodontal surgery.

AIM: To evaluate the protein profiles in gingival crevicular fluid (GCF) in relation to clinical outcomes after periodontal surgery and examine if any selected proteins affect the mRNA expression of pro-inflammatory cytokines in human gingival fibroblasts. MATERIALS AND METHODS: This exploratory study included 21 consecutive patients with periodontitis. GCF was collected, and the protein pattern (n = 92) and clinical parameters were evaluated prior to surgery and 3, 6 and 12 months after surgery. Fibroblastic gene expression was analysed by real-time quantitative polymerase chain reaction. RESULTS: Surgical treatment reduced periodontal pocket depth (PPD) and changed the GCF protein pattern. Twelve months after surgery, 17% of the pockets showed an increase in PPD. Levels of a number of proteins in the GCF decreased after surgical treatment but increased with early signs of tissue destruction, with LIGHT being one of the proteins that showed the strongest association. Furthermore, LIGHT up-regulated the mRNA expression of pro-inflammatory cytokines interleukin (IL)-6, IL-8 and MMP9 in human gingival fibroblasts. CONCLUSIONS: LIGHT can potentially detect subjects at high risk of periodontitis recurrence after surgical treatment. Moreover, LIGHT induces the expression of inflammatory cytokines and tissue-degrading enzymes in gingival fibroblasts.

Effects of bile salt-stimulated lipase on blood cells and associations with disease activity in human inflammatory joint disorders.

The bile salt-stimulated lipase (BSSL) was originally recognized as a lipolytic enzyme expressed by the exocrine pancreas and in some species, notably humans, the lactating mammary gland, being secreted into the duodenum and with the mother's milk, respectively. However, BSSL is also present in the blood and has been assigned additional functions, even beyond the gastrointestinal tract. Conventional BSSL knockout mice are protected from developing disease in animal models of arthritis, and antibodies directed towards BSSL prevent or mitigate disease in similar models. The aim of this study was to investigate the role of BSSL as a newly discovered player in inflammation and specifically in inflammatory joint disorders. As part of mechanism of action, we here show that BSSL is secreted by neutrophils, interacts with monocytes and stimulates their migration in vitro. An anti-BSSL antibody that blocks the human BSSL-monocyte interaction was shown to simultaneously prevent the signaling pathway by which BSSL induce cell migration. Moreover, in this cohort study we show that BSSL levels are significantly higher in blood samples from patients with rheumatoid arthritis and psoriatic arthritis compared to healthy controls. The BSSL levels in patients' blood also correlated with disease activity scores and established inflammatory markers. Hence, although the mode of action is not yet fully clarified, we conclude that BSSL could be considered a proinflammatory component in the innate immune system and thus a possible novel target for treatment of chronic inflammation.

Enamel matrix derivative does not affect osteoclast formation or bone resorption in cultures of mouse bone marrow macrophages or human monocytes.

OBJECTIVE: Enamel matrix derivative (EMD) is widely used under the brand name Emdogain® to promote periodontal regeneration in surgical treatment of periodontitis and peri-implantitis. The molecular mechanisms are unclear, but it has been proposed that EMD has stimulatory effects on the root cementum and periodontal ligament cells. Since dental implants lack these structures, we hypothesized that EMD-induced bone gain involve interactions with osteoclast precursor cells, with consequent inhibitory effect on osteoclast formation and/or activity. The aim was to evaluate this hypothesis. MATERIAL AND METHODS: Primary mouse bone marrow macrophages (BMMs) and human peripheral blood monocytes were cultured in the presence of receptor activator nuclear factor-κB ligand (RANKL) to stimulate osteoclast formation. A purified Emdogain® fraction was added to the cell cultures and the effect on number and size of newly formed osteoclasts were evaluated. In cultures on natural bone slices, bioanalytical methods were used to assay osteoclast number and bone resorption. RESULTS: EMD had a negative effect on osteoclastogenesis in mouse cultures on plastic surface, whereas addition of EMD to osteoclast precursor cells on bone substrate did not affect osteoclast formation or bone resorption. CONCLUSIONS: The results on natural bone matrix contradict a direct effect of EMD on osteoclast precursor cells.

Serum proteins associated with periodontitis relapse post-surgery: A pilot study.

BACKGROUND: The knowledge of which genes and proteins that are connected to the susceptibility to gingivitis with subsequent local tissue degradation seen in periodontitis is insufficient. Changes of serum proteins associated with recurrence of bleeding on probing (BOP) and increased periodontal pocket depths (PPD) after surgical treatment of periodontitis could reveal molecules that could be early signals of tissue destruction and/or of importance for systemic effects in other tissues or organs. METHODS: We performed a longitudinal pilot study and followed 96 inflammation-related proteins over time in serum from patients who underwent surgical treatment of periodontitis (n= 21). The samples were taken before (time 0), and then at 3, 6, and 12 months after surgery. Changes in protein levels were analysed in relation to the clinical outcome measures, that is, proportion of surfaces affected by BOP and PPD. RESULTS: Changes in treatment outcomes with early signs of relapse in periodontitis after surgical treatment, for example, increased BOP and PPDs, were during 12-months follow up associated with increased serum levels of high-sensitivity C-reactive protein (hs-CRP) and programmed death-ligand 1 (PD-L1), and reduced serum levels of cystatin-D protein. CONCLUSION: This study shows that clinical signs of recurrence of periodontitis after surgery are reflected in serum, but larger studies are needed for verification. Our novel findings of an association between increased PD-L1- and decreased cystatin D-levels and recurrence in periodontitis are interesting because PD-L1 has been shown to facilitate bacterial infections and chronic inflammation and cystatin D to inhibit tissue destruction. Our results justify mechanistic studies regarding the role of these molecules in periodontitis.

CCR3 deficiency is associated with increased osteoclast activity and reduced cortical bone volume in adult male mice.

Increasing evidence emphasizes the importance of chemokines and chemokine receptors as regulators of bone remodeling. The C-C chemokine receptor 3 (CCR3) is dramatically upregulated during osteoclastogenesis, but the role of CCR3 in osteoclast formation and bone remodeling in adult mice is unknown. Herein, we used bone marrow macrophages derived from adult male CCR3-proficient and CCR3-deficient mice to study the role of CCR3 in osteoclast formation and activity. CCR3 deficiency was associated with formation of giant hypernucleated osteoclasts, enhanced bone resorption when cultured on bone slices, and altered mRNA expression of related chemokine receptors and ligands. In addition, primary mouse calvarial osteoblasts isolated from CCR3-deficient mice showed increased mRNA expression of the osteoclast activator-related gene, receptor activator of nuclear factor kappa-B ligand, and osteoblast differentiation-associated genes. Microcomputed tomography analyses of femurs from CCR3-deficient mice revealed a bone phenotype that entailed less cortical thickness and volume. Consistent with our in vitro studies, the total number of osteoclasts did not differ between the genotypes in vivo. Moreover, an increased endocortical osteoid mineralization rate and higher trabecular and cortical bone formation rate was displayed in CCR3-deficient mice. Collectively, our data show that CCR3 deficiency influences osteoblast and osteoclast differentiation and that it is associated with thinner cortical bone in adult male mice.

Purification and characterization of recombinant human bile salt-stimulated lipase expressed in milk of transgenic cloned cows.

Bile salt-stimulated lipase (BSSL) is a lipolytic digestive enzyme with broad substrate specificity secreted from exocrine pancreas into the intestinal lumen in all species and from the lactating mammary gland into the milk of some species, notably humans but not cows. BSSL in breast milk facilitates digestion and absorption of milk fat and promotes growth of small for gestational age preterm infants. Thus, purified recombinant human BSSL (rhBSSL) can be used for treatment of patients with fat malabsorption and expressing rhBSSL in the milk of transgenic cloned cows would therefore be a mean to meet a medical need. In the present study, a vector pBAC-hLF-hBSSL was constructed, which efficiently expressed active rhBSSL in milk of transgenic cloned cows to a concentration of 9.8 mg/ml. The rhBSSL purified from cow milk had the same enzymatic activity, N-terminal amino acid sequence, amino acid composition and isoelectric point and similar physicochemical characteristics as human native BSSL. Our study supports the use of transgenic cattle for the cost-competitive, large-scale production of therapeutic rhBSSL.

Analysis of neurotrophic factors in limb and extraocular muscles of mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is currently an incurable fatal motor neuron syndrome characterized by progressive weakness, muscle wasting and death ensuing 3-5 years after diagnosis. Neurotrophic factors (NTFs) are known to be important in both nervous system development and maintenance. However, the attempt to translate the potential of NTFs into the therapeutic options remains limited despite substantial number of approaches, which have been tested clinically. Using quantitative RT-PCR (qRT-PCR) technique, the present study investigated mRNA expression of four different NTFs: brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4) and glial cell line-derived neurotrophic factor (GDNF) in limb muscles and extraocular muscles (EOMs) from SOD1G93A transgenic mice at early and terminal stages of ALS. General morphological examination revealed that muscle fibres were well preserved in both limb muscles and EOMs in early stage ALS mice. However, in terminal ALS mice, most muscle fibres were either atrophied or hypertrophied in limb muscles but unaffected in EOMs. qRT-PCR analysis showed that in early stage ALS mice, NT-4 was significantly down-regulated in limb muscles whereas NT-3 and GDNF were markedly up-regulated in EOMs. In terminal ALS mice, only GDNF was significantly up-regulated in limb muscles. We concluded that the early down-regulation of NT-4 in limb muscles is closely associated with muscle dystrophy and dysfunction at late stage, whereas the early up-regulations of GDNF and NT-3 in EOMs are closely associated with the relatively well-preserved muscle morphology at late stage. Collectively, the data suggested that comparing NTFs expression between limb muscles and EOMs from different stages of ALS animal models is a useful method in revealing the patho-physiology and progression of ALS, and eventually rescuing motor neuron in ALS patients.

Bile salt-stimulated lipase plays an unexpected role in arthritis development in rodents.

OBJECTIVE: The present study aimed to explore the hypothesis that bile salt-stimulated lipase (BSSL), in addition to being a key enzyme in dietary fat digestion during early infancy, plays an important role in inflammation, notably arthritis. METHODS: Collagen-induced arthritis (CIA) and pristane-induced arthritis (PIA) in rodents are commonly used experimental models that reproduce many of the pathogenic mechanisms of human rheumatoid arthritis, i.e. increased cellular infiltration, synovial hyperplasia, pannus formation, and erosion of cartilage and bone in the distal joints. We used the CIA model to compare the response in BSSL wild type (BSSL-WT) mice with BSSL-deficient 'knock-out' (BSSL-KO) and BSSL-heterozygous (BSSL-HET) littermates. We also investigated if intraperitoneal injection of BSSL-neutralizing antibodies affected the development or severity of CIA and PIA in mice and rats, respectively. RESULTS: In two consecutive studies, we found that BSSL-KO male mice, in contrast to BSSL-WT littermates, were significantly protected from developing arthritis. We also found that BSSL-HET mice were less prone to develop disease compared to BSSL-WT mice, but not as resistant as BSSL-KO mice, suggesting a gene-dose effect. Moreover, we found that BSSL-neutralizing antibody injection reduced both the incidence and severity of CIA and PIA in rodents. CONCLUSION: Our data strongly support BSSL as a key player in the inflammatory process, at least in rodents. It also suggests the possibility that BSSL-neutralizing agents could serve as a therapeutic model to reduce the inflammatory response in humans.

BSSL and PLRP2: key enzymes for lipid digestion in the newborn examined using the Caco-2 cell line.

In rodents, bile salt-stimulated lipase (BSSL) and pancreatic lipase-related protein 2 (PLRP2) are the dominant lipases expressed in the exocrine pancreas in early life when milk is the main food. The aim of the present study was to evaluate whether BSSL and PLRP2 are also key enzymes in neonatal intestinal fat digestion. Using Caco-2 cells as a model for the small intestinal epithelium, purified human enzymes were incubated in the apical compartment with substrates, bile salt composition and concentrations physiologic to newborn infants. Both BSSL and PLRP2 hydrolyzed triglycerides (TG) to free FA and glycerol. Released FA were absorbed by the cells and reesterfied to TG. Together, BSSL and PLRP2 had a synergistic effect, increasing cellular uptake and reesterification 4-fold compared with the sum of each lipase alone. A synergistic effect was also observed with retinyl ester as a substrate. PLRP2 hydrolyzed cholesteryl ester but not as efficiently as BSSL, and the two had an additive rather than synergistic effect. We conclude the key enzymes in intestinal fat digestion are different in newborns than later in life. Further studies are needed to fully understand this difference and its implication for designing optimal neonatal nutrition.

Diabetes and pancreatic exocrine dysfunction due to mutations in the carboxyl ester lipase gene-maturity onset diabetes of the young (CEL-MODY): a protein misfolding disease.

CEL-maturity onset diabetes of the young (MODY), diabetes with pancreatic lipomatosis and exocrine dysfunction, is due to dominant frameshift mutations in the acinar cell carboxyl ester lipase gene (CEL). As Cel knock-out mice do not express the phenotype and the mutant protein has an altered and intrinsically disordered tandem repeat domain, we hypothesized that the disease mechanism might involve a negative effect of the mutant protein. In silico analysis showed that the pI of the tandem repeat was markedly increased from pH 3.3 in wild-type (WT) to 11.8 in mutant (MUT) human CEL. By stably overexpressing CEL-WT and CEL-MUT in HEK293 cells, we found similar glycosylation, ubiquitination, constitutive secretion, and quality control of the two proteins. The CEL-MUT protein demonstrated, however, a high propensity to form aggregates found intracellularly and extracellularly. Different physicochemical properties of the intrinsically disordered tandem repeat domains of WT and MUT proteins may contribute to different short and long range interactions with the globular core domain and other macromolecules, including cell membranes. Thus, we propose that CEL-MODY is a protein misfolding disease caused by a negative gain-of-function effect of the mutant proteins in pancreatic tissues.

Binding of human milk to pathogen receptor DC-SIGN varies with bile salt-stimulated lipase (BSSL) gene polymorphism.

OBJECTIVE: Dendritic cells bind an array of antigens and DC-SIGN has been postulated to act as a receptor for mucosal pathogen transmission. Bile salt-stimulated lipase (BSSL) from human milk potently binds DC-SIGN and blocks DC-SIGN mediated trans-infection of CD4(+) T-lymphocytes with HIV-1. Objective was to study variation in DC-SIGN binding properties and the relation between DC-SIGN binding capacity of milk and BSSL gene polymorphisms. STUDY DESIGN: ELISA and PCR were used to study DC-SIGN binding properties and BSSL exon 11 size variation for human milk derived from 269 different mothers distributed over 4 geographical regions. RESULTS: DC-SIGN binding properties were highly variable for milks derived from different mothers and between samplings from different geographical regions. Differences in DC-SIGN binding were correlated with a genetic polymorphism in BSSL which is related to the number of 11 amino acid repeats at the C-terminus of the protein. CONCLUSION: The observed variation in DC-SIGN binding properties among milk samples may have implications for the risk of mucosal transmission of pathogens during breastfeeding.

Lipid digestion and absorption in early life: an update.

PURPOSE OF REVIEW: To highlight our understanding of digestion and absorption of dietary lipids in newborn infants, and specifically how these processes differ from those in children and adults. RECENT FINDINGS: The intestinal concentration of pancreatic triglyceride lipase (PTL) and bile salts is lower in newborns compared to later in life. Instead the PTL-related protein 2 and bile salt-stimulated lipase (BSSL) are the key enzymes secreted from pancreas, which in concerted action with gastric lipase operate to achieve efficient fat absorption during infancy. BSSL is also present in human milk which affects fat absorption and growth in breast-fed preterm infants. Under conditions of low luminal bile salt concentrations fat absorption is likely to occur from liquid crystalline product phases, which may result in absorption from an extended part of the small intestinal mucosal surfaces compared to adults. Chylomicron assembly and secretion also seem to adapt to the specific situation of the newborn. SUMMARY: Both fat digestion and product absorption are different in newborn infants compared to adults; other lipases are used for digestion and different physical-chemical phases may be used for product absorption. Why these differences occur is still an unsolved question of considerable importance to neonatal nutrition.

Adiponectin and peroxisome proliferator-activated receptor gamma expression in subcutaneous and omental adipose tissue in children.

AIM: To compare the expression levels of the adiponectin and peroxisome proliferator -activated receptor gamma (PPARgamma) genes in subcutaneous adipose tissue (SC) and omental adipose tissue (OM) in children with relation to age and anthropometric variables. METHODS: Paired biopsies (SC and OM) were obtained from 53 children (age 0.2-14 years, BMI 12.5-25.8 kg/m(2)). Adiponectin and PPARgamma mRNA levels in adipose tissue were measured by real-time PCR. RESULTS: In overweight, but not in normal weight children, the median adiponectin mRNA level was significantly lower in OM [0.51 (0.1-2.17)] compared to SC [1.29 (0.16-5.08)], (p = 0.03). Adiponectin mRNA levels were strongly associated with PPARgamma mRNA levels in both SC (r = 0.73, p < 0.001) and OM (r = 0.78, p < 0.001). CONCLUSIONS: The lower adiponectin expression in OM relative to SC in overweight children indicates that metabolic-endocrine alterations begin already in childhood. The close association between adiponectin and PPARgamma expression supports the hypothesis this transcription factor is involved in adiponectin gene regulation.

Bile salt-stimulated lipase and pancreatic lipase-related protein 2 are the dominating lipases in neonatal fat digestion in mice and rats.

During infancy, the basic conditions for digestion of dietary fat differ from later in life. The bile salt-stimulated lipase (BSSL) is an enzyme expressed in the exocrine pancreas and in some species (including human) also in the lactating mammary gland and secreted with the milk. The aim of this study was to compare the ontogeny of four pancreatic lipases [BSSL, pancreatic triglyceride lipase (PL), pancreatic lipase-related protein 2 (PLRP2), and phospholipase A2 (PLA2)] in one species that supplies BSSL with milk (the mouse) and one that does not (the rat). We followed expression of the four pancreatic lipases from postnatal d 1 until after weaning in both species. We found that BSSL and PLRP2, two lipases with broad substrate specificity, dominated. It was not until weaning that significant expression of PL and PLA2 were induced. Thus, BSSL and PLRP2 seem to be responsible for fat digestion as long as milk is the main food. Moreover, the early temporal pattern of BSSL expression differed between species. We speculate that the milk-borne BSSL is able to compensate for a slower ontogeny of pancreatic BSSL expression in the mouse.

Bile salt-stimulated lipase from human milk binds DC-SIGN and inhibits human immunodeficiency virus type 1 transfer to CD4+ T cells.

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium, Leishmania, and Helicobacter pylori, can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4+ T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric LeX component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (LeX)-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4+ T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in LeX expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.

Human bile salt-stimulated lipase has a high frequency of size variation due to a hypervariable region in exon 11.

The apparent molecular mass of human milk bile salt-stimulated lipase (BSSL) varies between mothers. The molecular basis for this is unknown, but indirect evidence has suggested the differences to reside in a region of repeats located in the C-terminal part of the protein. We here report that a polymorphism within exon 11 of the BSSL gene is the explanation for the molecular variants of BSSL found in milk. By Southern blot hybridization we analyzed the BSSL gene from mothers known to have BSSL of different molecular masses in their milk. A polymorphism was found within exon 11, previously shown to consist of 16 near identical repeats of 33 bp each. We detected deletions or, in one case, an insertion corresponding to the variation in molecular mass of the BSSL protein found in milk from the respective woman. Furthermore, we found that 56%, out of 295 individuals studied, carry deletions or insertions within exon 11 in one or both alleles of the BSSL gene. Hence, this is a hypervariable region and the current understanding that exon 11 in the human BSSL gene encodes 16 repeats is an oversimplification and needs to be revisited. Natural variation in the molecular mass of BSSL may have clinical implications.