Facile generation of monoclonal antibodies suitable for conjugation.

Monoclonal antibodies (mAb) are unique tools in therapeutics and immunodiagnostics applications but many of these applications rely on conjugated mAbs. Whether conjugating drugs or tracers, the conjugation process, frequently taking advantage of primary amines on lysine residues, may affect the binding activity of the antibodies. Furthermore, due to the sticky nature of many mAbs, unfavorable interactions may become eminent, with the result of high background signals. The workload associated with producing mAbs, able to withstand conjugation, preserving stability and affinity and avoiding off-target interactions, is comprehensive and related with only incidental success. We designed a method, where uncloned hybridomas were pre-selected for secretion of mAbs with the above characteristics. Using human collectin K1 (CL-K1, alias CL-11, Colec11) as a model antigen, mAbs present in culture supernatant from uncloned hybridomas were immobilized on Protein A beads, followed by solid phase biotinylation and subsequent elution. ELISA was employed to compare the binding activity of conjugated vs. unconjugated mAbs, and furthermore for their application in combination with other antibodies. From a group of 96 uncloned hybridomas we accomplished in obtaining five suitable mAbs, among which, two mAbs were superior. The successful conjugation of the selected mAbs with fluorophores and subsequent applications in microscopy and flow cytometry were further demonstrated. In conclusion, pre-selection of uncloned hybridomas, by testing of their mAbs' ability to withstand conjugation with tracers or drugs, is a successful strategy to avoid a huge workload of cloning numerous hybridomas, in order to obtain conjugatable mAbs.

Soluble pattern recognition molecules: Guardians and regulators of homeostasis at airway mucosal surfaces.

Maintenance of homeostasis at body barriers that are constantly challenged by microbes, toxins and potentially bioactive (macro)molecules requires complex, highly orchestrated mechanisms of protection. Recent discoveries in respiratory research have shed light on the unprecedented role of airway epithelial cells (AEC), which, besides immune cells homing to the lung, also significantly contribute to host defence by expressing membrane-bound and soluble pattern recognition receptors (sPRR). Recent evidence suggests that distinct, evolutionary ancient, sPRR secreted by AEC might become activated by usually innocuous proteins, commonly referred to as allergens. We here provide a systematic overview on sPRR detectable in the mucus lining of AEC. Some of them become actively produced and secreted by AECs (like the pentraxins C-reactive protein and pentraxin 3; the collectins mannose binding protein and surfactant proteins A and D; H-ficolin; serum amyloid A; and the complement components C3 and C5). Others are elaborated by innate and adaptive immune cells such as monocytes/macrophages and T cells (like the pentraxins C-reactive protein and pentraxin 3; L-ficolin; serum amyloid A; and the complement components C3 and C5). Herein we discuss how sPRRs may contribute to homeostasis but sometimes also to overt disease (e.g. airway hyperreactivity and asthma) at the alveolar-air interface.

Collectins: Innate Immune Pattern Recognition Molecules.

Collectins are collagen-containing C-type (calcium-dependent) lectins which are important pathogen pattern recognising innate immune molecules. Their primary structure is characterised by an N-terminal, triple-helical collagenous region made up of Gly-X-Y repeats, an a-helical coiled-coil trimerising neck region, and a C-terminal C-type lectin or carbohydrate recognition domain (CRD). Further oligomerisation of this primary structure can give rise to more complex and multimeric structures that can be seen under electron microscope. Collectins can be found in serum as well as in a range of tissues at the mucosal surfaces. Mannanbinding lectin can activate the complement system while other members of the collectin family are extremely versatile in recognising a diverse range of pathogens via their CRDs and bring about effector functions designed at the clearance of invading pathogens. These mechanisms include opsonisation, enhancement of phagocytosis, triggering superoxidative burst and nitric oxide production. Collectins can also potentiate the adaptive immune response via antigen presenting cells such as macrophages and dendritic cells through modulation of cytokines and chemokines, thus they can act as a link between innate and adaptive immunity. This chapter describes the structure-function relationships of collectins, their diverse functions, and their interaction with viruses, bacteria, fungi and parasites.

Functional characterization of partial recombinant goat conglutinin: Its role as innate immunity marker and use as antigen in sandwich ELISA.

Conglutinin, a liver synthesized versatile innate immune marker consisting C-type lectin domain belongs to collectin superfamily of proteins. The protein, first detected in bovine serum as soluble pattern recognition receptor (PRR) has wide range of antimicrobial activities. In the present study, open reading frame (ORF) encoding neck and carbohydrate recognition domain (NCRD) of goat conglutinin gene ligated to the vector pRSET-A was expressed in E. coli BL-21(pLys) cells. The 27 kDa recombinant protein (rGCGN) purified by single step Ni+2 -NTA affinity chromatography was found to cross-react with recombinant anti-buffalo conglutinin antibody raised in poultry. Further, it displayed calcium-dependant sugar binding activity towards yeast mannan and calcium-independent binding activity towards LPS. The mannan binding activity of rGCGN was inhibited in the presence of N-acetyl-glucosamine because of higher affinity towards this sugar. The recombinant protein was found to stimulate production of superoxide ions and hydrogen peroxide in goat neutrophils, which are instrumental in stimulating phagocytic activity of cells. When used as antigen in Sandwich ELISA, straight line (Y = 0.299x + 0.067, R2 = 0.997) was observed within the concentration range of 200-1000 ng/100 µl of rGCGN. Using this equation, the native conglutinin concentration in goat sera was estimated to be 0.5-7.5 µg/ml. The results indicated that prokaryotically expressed functionally active rGCGN can be used as antigen to assess native serum conglutinin levels in Sandwich ELISA and as immunomodulator in therapeutic applications to sequester unwanted immune complexes from the circulation.

Soluble defense collagens: Sweeping up immune threats.

Soluble defense collagens form a group of secreted proteins that are primarily involved in host defense. All defense collagens contain a globular recognition domain contiguous to a collagen-like triple helical domain. They are oligomeric proteins, assembled in multiples of three subunits due to their collagen domains. Members of this group include collectins such as surfactant protein A and D (SP-A, SP-D), and mannan-binding lectin; C1q, the first component of the complement system; adiponectin; and ficolins. All are secreted to tissue cavities or serum. Soluble defense collagens are specialized to respond to infection, triggering the initiation of the complement cascade and/or enhancing phagocytosis of pathogens by macrophages. However, once inflammation is established, C1q, collectins, ficolins, or adiponectin can influence macrophage responses, thereby contributing to resolve the inflammation. In addition, some members of this group of proteins (SP-A, C1q, and adiponectin) modulate tissue-repair functions of macrophages. This review will focus on the molecular mechanisms by which these proteins efficiently defend against immune threats and contribute to tissue repair.

Surfactant Proteins A and D: Trimerized Innate Immunity Proteins with an Affinity for Viral Fusion Proteins.

Innate recognition of viruses is an essential part of the immune response to viral pathogens. This is integral to the maintenance of healthy lungs, which are free from infection and efficient at gaseous exchange. An important component of innate immunity for identifying viruses is the family of C-type collagen-containing lectins, also known as collectins. These secreted, soluble proteins are pattern recognition receptors (PRRs) which recognise pathogen-associated molecular patterns (PAMPs), including viral glycoproteins. These innate immune proteins are composed of trimerized units which oligomerise into higher-order structures and facilitate the clearance of viral pathogens through multiple mechanisms. Similarly, many viral surface proteins form trimeric configurations, despite not showing primary protein sequence similarities across the virus classes and families to which they belong. In this review, we discuss the role of the lung collectins, i.e., surfactant proteins A and D (SP-A and SP-D) in viral recognition. We focus particularly on the structural similarity and complementarity of these trimeric collectins with the trimeric viral fusion proteins with which, we hypothesise, they have elegantly co-evolved. Recombinant versions of these innate immune proteins may have therapeutic potential in a range of infectious and inflammatory lung diseases including anti-viral therapeutics.

The Role of Complement Activating Collectins and Associated Serine Proteases in Patients With Hematological Malignancies, Receiving High-Dose Chemotherapy, and Autologous Hematopoietic Stem Cell Transplantations (Auto-HSCT).

We conducted a prospective study of 312 patients (194 with multiple myeloma, 118 with lymphomas) receiving high-dose conditioning chemotherapy and autologous hematopoietic stem cell transplantation (auto-HSCT). Polymorphisms of MBL2 and MASP2 genes were investigated and serial measurements of serum concentrations of mannose-binding lectin (MBL), CL-LK collectin and MASP-2 as well as activities of MBL-MASP-1 and MBL-MASP-2 complex were made. Serum samples were taken before conditioning chemotherapy, before HSCT and once weekly after (totally 4-5 samples); in minority of subjects also 1 and/or 3 months post transplantation. The results were compared with data from 267 healthy controls and analyzed in relation to clinical data to explore possible associations with cancer and with chemotherapy-induced medical complications. We found a higher frequency of MBL deficiency-associated genotypes (LXA/O or O/O) among multiple myeloma patients compared with controls. It was however not associated with hospital infections or post-HSCT recovery of leukocytes, but seemed to be associated with the most severe infections during follow-up. Paradoxically, high MBL serum levels were a risk factor for prolonged fever and some infections. The first possible association of MBL2 gene 3'-untranslated region polymorphism with cancer (lymphoma) in Caucasians was noted. Heterozygosity for MASP2 gene +359 A>G mutation was relatively frequent in lymphoma patients who experienced bacteremia during hospital stay. The median concentration of CL-LK was higher in myeloma patients compared with healthy subjects. Chemotherapy induced marked increases in serum MBL and MASP-2 concentrations, prolonged for several weeks and relatively slighter decline in CL-LK level within 1 week. Conflicting findings on the influence of MBL on infections following chemotherapy of myeloma and lymphoma have been reported. Here we found no evidence for an association between MBL deficiency and infection during the short period of neutropenia following conditioning treatment before HSCT. However, we noted a possible protective effect of MBL during follow-up, and suspected that to be fully effective when able to act in combination with phagocytic cells after their recovery.

Development of a Quantitative Assay for the Characterization of Human Collectin-11 (CL-11, CL-K1).

Collectin-11 (CL-11) is a pattern recognition molecule of the lectin pathway of complement with diverse functions spanning from host defense to embryonic development. CL-11 is found in the circulation in heterocomplexes with the homologous collectin-10 (CL-10). Abnormal CL-11 plasma levels are associated with the presence of disseminated intravascular coagulation, urinary schistosomiasis, and congenital disorders. Although there has been a marked development in the characterization of CL-11 there is still a scarcity of clinical tools for its analysis. Thus, we generated monoclonal antibodies and developed a quantitative ELISA to measure CL-11 in the circulation. The antibodies were screened against recombinant CL-11 and validated by ELISA and immunoprecipitation of serum and plasma. The best candidates were pairwise compared to develop a quantitative ELISA. The assay was validated regarding its sensitivity, reproducibility, and dilution linearity, demonstrating a satisfactory variability over a working range of 0.29-18.75 ng/ml. The mean plasma concentration of CL-11 in healthy controls was determined to be 289.4 ng/ml (range 143.2-459.4 ng/ml), highly correlated to the levels of CL/10/11 complexes (r = 0.729). Plasma CL-11 and CL-10/11 co-migrated in size exclusion chromatography as two major complexes of ~400 and >600 kDa. Furthermore, we observed a significant decrease at admission in CL-11 plasma levels in patients admitted to intensive care with systemic inflammatory response syndrome. By using the in-house antibodies and recombinant CL-11, we found that CL-11 can bind to zymosan independently of calcium by a separate site from the carbohydrate-binding region. Finally, we showed that CL-11/MASP-2 complexes trigger C4b deposition on zymosan. In conclusion, we have developed a specific and sensitive ELISA to investigate the ever-expanding roles of CL-11 in health and disease and shown a novel interaction between CL-11 and zymosan.

Novel Monoclonal Antibodies Against Native Human Collectin L1, Using the Heteromeric Complex CL-LK as Immunogen.

Collectin LK (CL-LK) is a recently described collectin complex, which upon binding to microbial glycoconjugates, activates the lectin pathway of the complement system, and thereby contributes to the removal of invading microorganisms. The complex consists of the two collectins; Collectin K1 (kidney 1 alias CL-11) and Collectin L1 (liver 1 alias CL-10). At present, most efforts have been made on the characterization of CL-K1, and little is known about the function of CL-L1 and its association with diseases. Deficiency of either of the two collectins is associated with the developmental syndrome 3MC, whereas increased plasma levels of CL-K1 are associated with disseminated intravascular coagulation. Using CL-LK purified from human plasma as an immunogen, we succeed in generating seven monoclonal antibodies (mAbs) with specificity for CL-L1. All seven mAbs recognize both native and recombinant CL-L1. In addition, four of the mAbs were successful in immunohistochemical detection of CL-L1 in human tissues. To our knowledge, these are the first mAbs specific for human native CL-L1 described in the literature, and we expect them to be of great importance in characterizing the function of CL-L1, as well as for the study of CL-L1's association with disease.

Expression and functional characterization of collection-K1 from Nile tilapia (Oreochromis niloticus) in host innate immune defense.

Collectin-K1 (CL-K1), a multifunctional Ca2+-dependent lectin, is able to bind carbohydrates on pathogens and inhibit infection by direct neutralization, agglutination, opsonization and killing, which plays an important role in innate immunity. In this study, a CL-K1 homolog (OnCL-K1) was identified from Nile tilapia (Oreochromis niloticus) and characterized at expression and agglutination functional levels. The open reading frame of OnCL-K1 is 720 bp of nucleotide sequence encoding a polypeptide of 239 amino acids. The deduced amino acid sequence has two characteristic structures, containing a collagen-like region and a carbohydrate recognition domain. Expression analysis revealed that the OnCL-K1 was highly expressed in the liver, and widely exhibited in other tissues including kidney, intestine and spleen. In addition, the OnCL-K1 expression was significantly up-regulated in spleen and anterior kidney following challenges with a Gram-positive bacterial pathogen (Streptococcus agalactiae) and a Gram-negative bacterial pathogen (Aeromonas hydrophila). The up-regulation of OnCL-K1 expression was also demonstrated in hepatocytes and monocytes/macrophages in vitro stimulation with S. agalactiae and A. hydrophila. Recombinant OnCL-K1 protein was able to agglutinate both S. agalactiae and A. hydrophila in vitro, and participate in the regulation of inflammatory, migration reaction and promote the phagocytosis by monocytes/macrophages. Taken together, the results of this study indicated that OnCL-K1, possessing apparent agglutination, opsonization and killing ability to bacterial pathogens and participating in the regulation mechanisms of the non-specific cellular immune, might be involved in host defense of innate immunity against bacterial infection in Nile tilapia.

Identification of genetic variation in equine collagenous lectins using targeted resequencing.

Collagenous lectins are a family of soluble pattern recognition receptors that play an important role in innate immune resistance to infectious disease. Through recognition of carbohydrate motifs on the surface of pathogens, some collagenous lectins can activate the lectin pathway of complement, providing an effective means of host defense. Genetic polymorphisms in collagenous lectins have been shown in several species to predispose animals to a variety of infectious diseases. Infectious diseases are an important cause of morbidity in horses, however little is known regarding the role of equine collagenous lectins. Using a high-throughput, targeted re-sequencing approach, the relationship between genetic variation in equine collagenous lectin genes and susceptibility to disease was investigated. DNA was isolated from tissues obtained from horses submitted for post-mortem examination. Animals were divided into two populations, those with infectious or autoinflammatory diseases (n = 37) and those without (n = 52), and then subdivided by dominant pathological process for a total of 21 pools, each containing 4-5 horses. DNA was extracted from each horse and pooled in equimolar amounts, and the exons, introns, upstream (approximately 50 kb) and downstream (approximately 3 kb) regulatory regions for the 11 equine collagenous lectin genes and related MASP genes were targeted for re-sequencing. A custom target capture kit was used to prepare a sequencing library, which was sequenced on an Illumina MiSeq. After implementing quality control filters, 4559 variants were identified. Of these, 92 were present in the coding regions (43 missense, 1 nonsense, and 48 synonymous), 1414 in introns, 3029 in the upstream region, and 240 in the downstream region. In silico analysis of the missense short nucleotide variants identified 12 mutations with potential to disrupt collagenous lectin protein structure or function, 280 mutations located within predicted transcription factor binding sites, and 95 mutations located within predicted microRNA binding elements. Analysis of allelic association identified 113 mutations that segregated between the infectious/autoinflammatory and non-infectious populations. The variants discovered in this experiment represent potential genetic contributors to disease susceptibility of horses, and will serve as candidates for further population-level genotyping. This study contributes to the growing body of evidence that pooled, high-throughput sequencing is a viable strategy for cost-effective variant discovery.

CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa.

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.

Complement Dependent and Independent Interaction Between Bovine Conglutinin and Mycobacterium bovis BCG: Implications in Bovine Tuberculosis.

Bovine conglutinin, the first animal collectin to be discovered, is structurally very similar to Surfactant Protein D (SP-D). SP-D is known to interact with Mycobacterium tuberculosis, and the closely-related M. bovis, the causative agent of bovine tuberculosis. We speculated that due to the overall similarities between conglutinin and SP-D, conglutinin is likely to have a protective influence in bovine tuberculosis. We set out to investigate the role of conglutinin in host-pathogen interaction during mycobacterial infection. We show here that a recombinant truncated form of conglutinin (rfBC), composed of the neck and C-type lectin domains, binds specifically and in a dose-dependent manner to the model organism Mycobacterium bovis BCG. rfBC showed a significant direct bacteriostatic effect on the growth of M. bovis BCG in culture. In addition, rfBC inhibited the uptake of M. bovis BCG by THP-1 macrophages (human monocyte lineage cell line) and suppressed the subsequent pro-inflammatory response. Conglutinin is well-known as a binder of the complement activation product, iC3b. rfBC was also able to inhibit the uptake of complement-coated M. bovis BCG by THP-1 macrophages, whilst modulating the pro-inflammatory response. It is likely that rfBC inhibits the phagocytosis of mycobacteria by two distinct mechanisms: firstly, rfBC interferes with mannose receptor-mediated uptake by masking lipoarabinomannan (LAM) on the mycobacterial surface. Secondly, since conglutinin binds iC3b, it can interfere with complement receptor-mediated uptake via CR3 and CR4, by masking interactions with iC3b deposited on the mycobacterial surface. rfBC was also able to modulate the downstream pro-inflammatory response in THP-1 cells, which is important for mobilizing the adaptive immune response, facilitating containment of mycobacterial infection. In conclusion, we show that conglutinin possesses complement-dependent and complement-independent anti-mycobacterial activities, interfering with both known mechanisms of mycobacterial uptake by macrophages. As mycobacteria are specialized intracellular pathogens, conglutinin may inhibit M. bovis and M. tuberculosis from establishing an intracellular niche within macrophages, and thus, negatively affect the long-term survival of the pathogen in the host.

Collectins and galectins in the abomasum of goats susceptible and resistant to gastrointestinal nematode infection.

Originally described in cattle, conglutinin belongs to the collectin family and is involved in innate immune defense. It is thought that conglutinin provides the first line of defense by maintaining a symbiotic relationship with the microbes in the rumen while inhibiting inflammatory reactions caused by antibodies leaking into the bloodstream. Due to the lack of information on the similar lectins and sequence detection in goats, we characterized the goat conglutinin gene using RACE and evaluated the differences in its gene expression profile, as well as in the gene expression profiles for surfactant protein A, galectins 14 and 11, interleukin 4 and interferon-gamma in goats. We used Saanen and Anglo Nubian F2 crossbred goats monitored over a period of four months and characterized them as resistant (R) or susceptible (S) based on the average values of EPG counts. Goat conglutinin was similar to bovine conglutinin, but its gene expression varied among different tissues. However, as with bovine conglutinin, it was most highly expressed in the liver. Variation in conglutinin (R=24.3±3.9; S=23.5±2.6, p=0.059), protein surfactant A (R=23.8±5.2, S=24.4±2.3, p=0.16), galectin 14 (R=15.9±3.5, S=14.7±6.2, p=0.49) and galectin l1 gene expression (R=25.4±2.6, S=25.8±3.7, p=0.53) was not significant between groups. However, there were weak correlations between interleukin 4 and the protein surfactant A gene (r=0.459, p=0.02) and between interleukin 4 and galectin 11 (r=0.498, p=0.01). Strong correlation between interferon-gamma and galectin 14 (r=0.744, p=0.00) was observed. Galectin 14 was negatively correlated with the number of nematodes in the goat (r=-0.416, p=0.04) as well as the EPG count (r=-0.408, p=0.04). This is the first study to date that identifies the gene expression of conglutinin, surfactant protein A and galectins 14 and 11 in the goat abomasum. In conclusion, we present evidence that lectin is involved in the immune response to gastrointestinal nematodes, which suggests that collectins and galectins are involved in the molecular recognition of helminths.

Collectins in urinary tract and kidney diseases.

The innate immune system serves as the frontline defense against invading pathogens and initiates an inflammatory response to microorganisms. Collectins are C-type lectins that are structurally characterized by a collagen-like sequence and a carbohydrate recognition domain. Moreover, they are widely expressed throughout the body and are involved in the innate immunity against a variety of pathogens, regulating inflammation, and protecting the lungs from pathogens. Recently, two classical collectins, surfactant protein A (SP-A) and surfactant protein D (SP-D), as well as novel collectin 11, were found present in urinary tract tissues. They are increasingly recognized as key players in activating the humoral arm of innate immunity and host defense in urinary tract and kidney diseases, although their biological features, functions, and mechanisms in this regard remain largely unclear. In this review, we aim to integrate results reported by ourselves and others to summarize and gain a better understanding of the functions of collectins (SP-A, SP-D, and collectin 11) in urinary tract and kidney diseases.

Complement Recognition Pathways in Renal Transplantation.

The complement system, consisting of soluble and cell membrane-bound components of the innate immune system, has defined roles in the pathophysiology of renal allograft rejection. Notably, the unavoidable ischemia-reperfusion injury inherent to transplantation is mediated through the terminal complement activation products C5a and C5b-9. Furthermore, biologically active fragments C3a and C5a, produced during complement activation, can modulate both antigen presentation and T cell priming, ultimately leading to allograft rejection. Earlier work identified renal tubule cell synthesis of C3, rather than hepatic synthesis of C3, as the primary source of C3 driving these effects. Recent efforts have focused on identifying the local triggers of complement activation. Collectin-11, a soluble C-type lectin expressed in renal tissue, has been implicated as an important trigger of complement activation in renal tissue. In particular, collectin-11 has been shown to engage L-fucose at sites of ischemic stress, activating the lectin complement pathway and directing the innate immune response to the distressed renal tubule. The interface between collectin-11 and L-fucose, in both the recipient and the allograft, is an attractive target for therapies intended to curtail renal inflammation in the acute phase.

An overview of the synergy and crosstalk between pentraxins and collectins/ficolins: their functional relevance in complement activation.

The complement system is an innate immune defense machinery comprising components that deploy rapid immune responses and provide efficient protection against foreign invaders and unwanted host elements. The complement system is activated upon recognition of pathogenic microorganisms or altered self-cells by exclusive pattern recognition molecules (PRMs), such as collectins, ficolins and pentraxins. Recent accumulating evidence shows that the different classes of effector PRMs build up a co-operative network and exert synergistic effects on complement activation. In this review, we describe our updated view of the crosstalk between previously unlinked PRMs in complement activation and the potential pathogenic effects during infection and inflammation.

[Diagnostic significance of anti-collectin 11 in systemic lupus erythematosus].

OBJECTIVE: To analyze the role of anti-collectin 11 in the diagnosis of systemic lupus erythematosus (SLE) and in the evaluation of disease activity. METHODS: This was a cross-sectional study. Five groups of patients were enrolled: SLE active (SLE disease activity index-2000,SLEDAI-2000≥9), SLE remission (SLEDAI-2000≤4 and there was no organ involvement), rheumatoid arthritis (RA), primary Sjogren Syndrome (SS) and healthy control (HC). Serum anti-collectin 11 was detected in all the groups by ELISA. One-way ANOVA analysis and LSD-t-test as post-hoc analysis were used to compare the levels of anti-collectin 11 among all the groups. Receiver operating characteristic (ROC) curve and the area under curve (AUC) were used to analyze the value of anti-collectin 11 in the diagnosis of SLE. RESULTS: In the study, 30 patients were enrolled in each group, including 13 males and 137 females with an average age of (34±14) years (18-77 years). The age and gender of the other three groups were comparable to the two SLE groups. The difference of serum anti-collectin 11 between the SLE active group and SLE remission group was not statistically significant (88.8±16.8 vs. 89.7±24.7, P=0.896). The level of serum anti-collectin 11 was significantly higher in SLE group (as a whole) (89.1±19.4) than in RA group (49.1±22.0), SS group (56.9±30.1) and HC group (72.7±24.6) (P<0.001, P<0.001, P=0.007, respectively). The AUC was 0.806 for the diagnosis of SLE by serum anti-collectin 11. Further descriptive analysis showed that the positive rate of anti-collectin 11 was very high in the patients of SLE in whom both anti-double-stranded DNA (dsDNA) and Sm antibody were negative. The nervous system and gastrointestinal system involvement were the most common in the patients with positive anti-collectin 11. CONCLUSION: The level of serum anti-collectin 11 was significantly higher in SLE than in RA, SS and HC. anti-collectin 11 antibody had a relatively high value in the diagnosis of SLE and it might have some complementary function in the diagnosis of SLE. It might be a relatively specific autoantibody for SLE.

Pentraxins and Collectins: Friend or Foe during Pathogen Invasion?

Innate immunity serves as the frontline defence against invading pathogens. Despite decades of research, new insights are constantly challenging our understanding of host-elicited immunity during microbial infections. Recently, two families of humoral innate immune proteins, pentraxins and collectins, have become a major focus of research in the field of innate immunity. Pentraxins and collectins are key players in activating the humoral arm of innate immunity, taking centre stage in immunoregulation and disease modulation. However, increasing evidence suggests that pentraxins and collectins can also mediate pathogenic effects during some infections. Herein, we discuss the protective and pathogenic effects of pentraxins and collectins, as well as their therapeutic significance.

Soluble Collectin-12 (CL-12) Is a Pattern Recognition Molecule Initiating Complement Activation via the Alternative Pathway.

Soluble defense collagens including the collectins play important roles in innate immunity. Recently, a new member of the collectin family named collectin-12 (CL-12 or CL-P1) has been identified. CL-12 is highly expressed in umbilical cord vascular endothelial cells as a transmembrane receptor and may recognize certain bacteria and fungi, leading to opsonophagocytosis. However, based on its structural and functional similarities with soluble collectins, we hypothesized the existence of a fluid-phase analog of CL-12 released from cells, which may function as a soluble pattern-recognition molecule. Using recombinant CL-12 full length or CL-12 extracellular domain, we determined the occurrence of soluble CL-12 shed from in vitro cultured cells. Western blot showed that soluble recombinant CL-12 migrated with a band corresponding to ∼ 120 kDa under reducing conditions, whereas under nonreducing conditions it presented multimeric assembly forms. Immunoprecipitation and Western blot analysis of human umbilical cord plasma enabled identification of a natural soluble form of CL-12 having an electrophoretic mobility pattern close to that of shed soluble recombinant CL-12. Soluble CL-12 could recognize Aspergillus fumigatus partially through the carbohydrate-recognition domain in a Ca(2+)-independent manner. This led to activation of the alternative pathway of complement exclusively via association with properdin on A. fumigatus as validated by detection of C3b deposition and formation of the terminal complement complex. These results demonstrate the existence of CL-12 in a soluble form and indicate a novel mechanism by which the alternative pathway of complement may be triggered directly by a soluble pattern-recognition molecule.