A Siglec-1-like lectin from Nile tilapia (Oreochromis niloticus) possesses functions of agglutination and mediation of macrophage phagocytic activity.

Siglec-1, one of the sialic acid-binding immunoglobulin-type lectins, is closely related to the recognition of host-pathogen and cell-cell interactions in the adaptive and innate immune systems. In this communication, a Siglec-1-like gene (OnSiglec-1-like) from Nile tilapia (Oreochromis niloticus) was analyzed. Relative expression revealed that the OnSiglec-1-like was expressed in all tested tissues, and the highest expression was found in the anterior kidney. Upon Streptococcus agalactiae (S. agalactiae) infection, the expression of OnSiglec-1-like was up-regulated in anterior kidney and spleen significantly in vivo. Additionally, the same phenomenon was observed in anterior kidney leukocytes upon LPS and S. agalactiae challenges as well in vitro. Western-blotting and ELISA analyses revealed that recombinant OnSiglec-1-like protein possessed high binding activity to LTA, LPS and S. agalactiae. Further, the recombinant OnSiglec-1-like was able to agglutinate S. agalactiae. Moreover, with the digestion of specific sialidase, the phagocytic ability of macrophages to S. agalactiae was greatly enhanced. Taken together, these results indicated that the Siglec-1-like possesses conserved functions of agglutination and promotion of macrophage phagocytic activity in Nile tilapia.

Insect C-Type Lectins in Microbial Infections.

C-type lectins (CTLs) are a family of carbohydrate-recognition domain (CRD)-containing proteins that bind to ligands in a calcium-dependent manner. CTLs act as important components of insect innate immune responses, such as pattern recognition, agglutination, encapsulation, melanization, phagocytosis and prophenoloxidase activation, as well as gut microbiome homeostasis maintenance, to defend against pathogens. Besides, some insect CTLs can facilitate pathogen infection and colonization. In this review, we describe the properties of insect CTLs and focus on explaining their role in viral, bacterial, parasitic and fungal infections.

Semi-Quantitative Impacts of Major Step on Antigen in Pre-Treatment Required for Immunohistochemistry.

BACKGROUND: We investigated the impact of immunohistochemistry (IHC) pre-treatment steps on antigens. METHODS: Salmonella typhimurium was selected as the observed antigen. The antigen was subjected to IHC pre-treatment steps involving a series of reagents, including 10% formaldehyde, ethanol, and xylene. Antigenicity was then measured by agglutination reaction. RESULTS: The agglutination titer for S. typhimurium was higher in the untreated control group than in the experimental group, indicating that pre-treatment inhibited antigen activity. The inhibitory effect of ethanol was greater than that of 10% formaldehyde and xylene. Unexpectedly, partial antigen recovery can be achieved from a preparation of paraffin section after hydration. CONCLUSIONS: S. Antigens may be strongly inhibited (inhibition: 70.8%) by IHC pre-treatment steps, especially by alcohol treatment. There is an experimental foundation for antigen retrieval in IHC.

Sialic acid-binding lectins (SABLs) from Solen grandis function as PRRs ensuring immune recognition and bacterial clearance.

Sialic acid-binding lectins (SABLs) are ubiquitous ancient molecules with binding properties to N-acetyl or N-glycolyl carbohydrates, and play crucial roles in both adaptive and innate immune responses. In present study, recombinant protein and antibodies of two SABLs from mollusk Solen grandis (SgSABL-1 and SgSABL-2) were prepared to investigate their functions in innate immunity. The recombinant protein of SgSABL-1 (rSgSABL-1) could bind LPS, PGN and ß-glucan in vitro, while rSgSABL-2 could only bind PGN rather than LPS and ß-glucan. Be coincident with their PAMPs recognition properties, rSgSABL-1 displayed a broad agglutination spectrum towards gram-positive bacteria Micrococcus luteus, gram-negative bacteria Listonella anguillarum and fungi Pichia pastoris, and rSgSABL-2 only showed remarkable agglutinative effect on M. luteus and L. anguillarum. More importantly, after PAMPs recognition, rSgSABL-1 and rSgSABL-2 enhanced phagocytosis as well as encapsulation ability of hemocytes in vitro, and the enhanced encapsulation could be blocked by specific antibodies. All these results indicated that SgSABL-1 and SgSABL-2 functioned as two compensative pattern-recognition receptor (PRRs) with distinct recognition spectrum and involved in the innate immune response of S. grandis.

A Rare Non-Hemolytic Case of Idiopathic Cold Agglutinin Disease.

BACKGROUND: Cold agglutinin disease is a very rare condition associated with agglutination of erythrocytes in cold environment usually due to IgM type antibodies. Other than hemolytic anemias, it may interfere with routine hemogram tests due to miscalculation of red blood cell count (RBC) and other hemogram parameters calculated with involvement of RBC. Awareness of the condition is important to overcome laboratory errors. METHODS: We studied a peripheral blood smear and repeated the hemogram test at 37°C to establish the diagnosis of cold agglutinin disease. RESULTS: Initial hemogram test results of the fifty-eight year-old man was as follows: RBC: 1.34 M/µL, hemoglobin (Hb): 12.4 g/dL, hematocrit (Htc): 11.8%, mean corpuscular hemoglobin (MCH): 92.4 pg, and mean corpuscular hemoglobin concentration (MCHC): 105 gr/dL. Despite the standard indirect Coombs test being negative, repeated tests at room temperature was 4+. We suspected cold agglutinin disease and repeated the hemogram test using the Bain-Marie method at 37°C and the test results showed RBC: 3.4 M/µL, hemoglobin: 12.6 g/dL, hematocrit: 30.2%, MCH: 31.7 pg, and MCHC: 41.8 g/dL. CONCLUSIONS: Inappropriate hemogram results may be a sign of underlying cold agglutinin disease. Hemolytic anemia not always accompanies the disease; however, cold exposure may trigger erythrocyte agglutination in vitro and may cause erratic laboratory results.

A novel C-type lectin in the black tiger shrimp Penaeus monodon functions as a pattern recognition receptor by binding and causing bacterial agglutination.

C-type lectins are pattern recognition proteins that play important roles in innate immunity in invertebrates by mediating the recognition of pathogens. In this study, a novel C-type lectin gene, PmCLec, was cloned and characterized from the black tiger shrimp Penaeus monodon. The open reading frame of PmCLec is 657 bp in length. It encodes a predicted protein of 218 amino acids with a calculated molecular mass and an isoelectric point of 24086 Da and 4.67, respectively. Sequence analysis of PmCLec showed similarity to members of the C-type lectin gene superfamily. The deduced protein contains a single carbohydrate recognition domain (CRD) and four conserved cysteine residues (Cys58, Cys126, Cys141, Cys149) that are involved in the formation of disulfide bridges. PmCLec transcripts are expressed in various tiger shrimp tissues, with the highest expression in the lymphoid organ. RNAi-mediated silencing of PmCLec resulted in higher cumulative mortality of knockdown shrimp after Vibrio harveyi infection compared to the control groups. Recombinant PmCLec was successfully expressed in the E. coli system. In the presence of Ca2+, purified rPmCLec protein binds and agglutinates Gram-positive bacteria (Staphylococcus aureus, S. hemolyticus), but only slightly binds and agglutinates E. coli and could not bind to the Gram-negative bacteria Bacillus megaterium and Vibrio harveyi. These results suggest that PmCLec functions as a pattern recognition receptor that is implicated in shrimp innate immunity.

Rapid effects of a protective O-polysaccharide-specific monoclonal IgA on Vibrio cholerae agglutination, motility, and surface morphology.

2D6 is a dimeric monoclonal immunoglobulin A (IgA) specific for the nonreducing terminal residue of Ogawa O-polysaccharide (OPS) of Vibrio cholerae. It was previously demonstrated that 2D6 IgA is sufficient to passively protect suckling mice from oral challenge with virulent V. cholerae O395. In this study, we sought to define the mechanism by which 2D6 IgA antibody protects the intestinal epithelium from V. cholerae infection. In a mouse ligated-ileal-loop assay, 2D6 IgA promoted V. cholerae agglutination in the intestinal lumen and limited the ability of the bacteria to associate with the epithelium, particularly within the crypt regions. In vitro fluorescence digital video microscopy analysis of antibody-treated V. cholerae in liquid medium revealed that 2D6 IgA not only induced the rapid (5- to 10-min) onset of agglutination but was an equally potent inhibitor of bacterial motility. Scanning electron microscopy showed that 2D6 IgA promoted flagellum-flagellum cross-linking, as well as flagellar entanglement with bacterial bodies, suggesting that motility arrest may be a consequence of flagellar tethering. However, monovalent 2D6 Fab fragments also inhibited V. cholerae motility, demonstrating that antibody-mediated agglutination and motility arrest are separate phenomena. While 2D6 IgA is neither bactericidal nor bacteriostatic, exposure of V. cholerae to 2D6 IgA (or Fab fragments) resulted in a 5-fold increase in surface-associated blebs, as well an onset of a wrinkled surface morphotype. We propose that the protective immunity conferred by 2D6 IgA is the result of multifactorial effects on V. cholerae, including agglutination, motility arrest, and possibly outer membrane stress.

HdhCTL1 is a novel C-type lectin of abalone Haliotis discus hannai that agglutinates Gram-negative bacterial pathogens.

C-type lectins (CTLs) are Ca(2+)-dependent carbohydrate recognition proteins, which play important roles in the innate immunity of both vertebrates and invertebrates. In this study, we identified and characterized a C-type lectin (named HdhCTL1) from Pacific abalone, Haliotis discus hannai. HdhCTL1 is composed of 176 amino acid residues and shares low (23.9%) identity with the known CTL of abalone. HdhCTL1 possesses a putative signal peptide and a carbohydrate-recognition domain (CRD) typical of CTLs. The CRD of HdhCTL1 contains four disulfide bond-forming cysteine residues that are highly conserved in CTLs. HdhCTL1 mRNA was detected in a wide range of tissues and expressed abundantly in the digestive gland. Experimental infection with the bacterial pathogen Vibrio anguillarum significantly upregulated HdhCTL1 expression in a time-dependent manner. Recombinant HdhCTL1 (rHdhCTL1) purified from Escherichia coli was able to agglutinate Gram-negative bacterial pathogens. The agglutinating ability of rHdhCTL1 was abolished in the presence of mannose. These results suggest that HdhCTL1 is a novel CTL which is likely to be involved in host defense against bacterial infection.

Natural defense by saliva and mucosa against oral infection by Leptospira.

Leptospirosis caused by drinking water has not been as frequently reported as percutaneous infection. Resistance to oral infection by pathogenic Leptospira was examined in an experimental hamster infection model. The results suggested some natural defenses against oral infection by Leptospira. First, we found that characteristic linear agglutination of Leptospira rapidly occurs when mixed with human saliva. That human saliva attenuated the infectivity of the treated leptospires by its agglutination activity suggested saliva to be the first line of defense against oral infection by leptospires. Second, only 10(1) Leptospira organisms caused death after submucosal injection into oral mucosa in hamsters, but oral infection with drinking water containing 10(5) organisms/mL did not cause death. This result showed that the mucosa plays the role of a physical barrier. Third, hamsters intragastrically infected by leptospires, with doses lethal to hamsters in oral infection, showed no signs of illness, which suggested that gastric acid plays an important role in preventing oral infection. Based on these results, saliva, mucosa, and gastric acid make up a natural defense, which confers high resistance to hosts against oral infection by leptospires.

[The development of polymer immunoglobulin preparations to identify different serovars legionella pneumophilia in reaction of slide-agglutination].

The article deals with the results of study targeted to develop polymer diagnostic preparation to identify epidemically significant serogroups Legionella pneumophilia. The preparation combines rate of record (1-5 min) of reaction of paragglutinining preparations with color visualization and demonstrative of reaction of volume agglomeration with polymer diagnosticums. The specially synthesized polymer microspheres were sensibilized with serums enriched with antibodies to lipopolysaccharide of corresponding serovar L. pneumophilia. The derived immunoglobulin diagnostic preparations detect agent of legionellesis in the reaction of slide-agglutination on glass during 1-5 min. The polymer diagnostic preparations provide positive reaction with culture of corresponding serovar and no reaction with other gomologic and geterologic agents of infectious diseases.

[The latex agglutination with video digital registration: the enhancement of diagnostic significance of conventional technique].

The rapid semiquantitative latex-tests, because of their analytic characteristics and convenient application, became widespread in the practice of laboratory diagnostics. Though, in spite of high sensitivity and specificity, their diagnostic effectiveness is lower that it could be mainly because of the impossibility to document the results of latex agglutinative re4actions and to manage the objective quality control. The application of systems of video digital registration permits to enhance the clinical significance of these analyses. By means of scanner systems (control and program complex "Expert Lab") the image of analytic objects is received with the results of latex agglutination reaction. The application of program techniques (the programs "Expert Lab - Agglutination" and "Expert Lab - Agglutination - Micros") in data processing permits to get the precise qualitative characteristics of active reactions, to ensure the automatic interpretation of results and gives an opportunity to proceed with the internal laboratory quality control. The saving of analytic object image in computer memory after termination of reaction favors the formation of data base, the implementation of retrospective evaluation of obtained results, additional consultations in dubious cases, including on-line. The application of complex "Expert Lab" permitted to develop the miniaturizes matrix systems permitting to decrease the withdrawal of latex reagents, to increase the productivity of analytical stage of operation preserving all analytical characteristics of method.

C-type lectin from red swamp crayfish Procambarus clarkii participates in cellular immune response.

Lectins are potential immune recognition proteins. In this study, a novel C-type lectin (Pc-Lec1) is reported in freshwater crayfish Procambarus clarkii. Pc-Lec1 encodes a protein of 163 amino acids with a putative signal peptide and a single carbohydrate recognition domain. It was constitutively expressed in various tissues of a normal crayfish, especially in the hepatopancreas and gills. Expressions of Pc-Lec1 were up-regulated in the hepatopancreas and gills of crayfish challenged with Vibrio anguillarum, Staphylococcus aureus, or the white spot syndrome virus. Recombinant mature Pc-Lec1 bound bacteria and polysaccharides (peptidoglycan, lipoteichoic acid, and lipopolysaccharide) but did not agglutinate bacteria. Pc-Lec1 enhanced hemocyte encapsulation of the sepharose beads in vitro, and the blocking of beads by a polyclonal antibody inhibited encapsulation. Pc-Lec1 promoted clearance of V. anguillarum in vivo. These results suggest that Pc-Lec1 is a pattern recognition receptor and participates in cellular immune response. Pc-Lec1 performs its function as an opsonin by enhancing the encapsulation or clearance of pathogenic bacteria.

Two fibrinogen-related proteins (FREPs) in the razor clam (Sinonovacula constricta) with a broad recognition spectrum and bacteria agglutination activity.

Fibrinogen-related proteins (FREPs) that contain only the fibrinogen-related domain are likely involved in pathogen recognition. In this study, we identified two FREPs from the razor clam (Sinonovacula constricta), called ScFREP-1 and ScFREP-2, and investigated their roles in the immune response. Both ScFREP-1 and ScFREP-2 contained a fibrinogen-related domain at the C-terminal. ScFREP-1 and ScFREP-2 mRNAs were detected in all adult clam tissues tested, with the highest expression levels in the gill and mantle, respectively. Their expression levels were significantly upregulated after microbe infection. Recombinant ScFREPs could bind Gram-positive and Gram-negative bacteria as well as some pathogen-associated molecular patterns (PAMPs), and they could agglutinate those bacteria. These results showed that ScFREPs functioned as potential pattern recognition receptors to mediate immune response by recognizing PAMPs and agglutinating invasive microbes.

Expanded collectin family in bullfrog (Rana catesbeiana): Identification and characterization of plasma collectins.

BLAST searches against databases for the bullfrog (Rana catesbeiana), using the collectin sequence previously identified in tadpoles, revealed the presence of at least 20 members of the collectin gene family. Phylogenetic analysis demonstrated that the bullfrog possesses expanded gene subfamilies encoding mannose-binding lectin (MBL) and pulmonary surfactant-associated protein D (PSAPD). Two collectins, of 20 kDa (PSAPD1) and 25 kDa (PSAPD6), were purified as a mixture from adult bullfrog plasma using affinity chromatography. These collectins were present as an oligomer of ~400 kDa in their native state, and showed Ca2+-dependent carbohydrate binding with different sugar preferences. Affinity-purified collectins showed weak E. coli agglutination and bactericidal activities, compared with those of plasma. Although both PSAPD1 and PSAPD6 genes were predominantly expressed in the liver, PSAPD1 transcripts were abundant in adults whereas PSAPD6 transcripts were abundant in tadpoles. The findings indicate that two gene subfamilies in the collectin family have diverged structurally, functionally and transcriptionally in the bullfrog. Rapid expansion of the collectin family in bullfrogs may reflect the onset of sub-functionalization of the prototype MBL gene towards tetrapod MBL and PSAPDs, and may be one means of natural adaptation in the innate immune system to various pathogens in both aquatic and terrestrial environments.

A C-type lectin (OmCTL) in Onychostoma macrolepis: Binding ability to LPS, PGN and agglutinating activity against bacteria.

C-type lectins (CTLs) are calcium-dependent carbohydrate-binding proteins that mainly bind to carbohydrate-based or other ligands to mediate cell adhesion, recognize pathogens, and play important roles in the immune system. In the present study, a novel C-type lectin (OmCTL) isolated from Onychostoma macrolepis was investigated. The open reading frame of OmCTL comprises 468 bp, encoding a 155 amino acid polypeptide with an 18 amino acid putative signaling peptide. The predicted primary OmCTL structure contains a signal peptide, a single carbohydrate recognition domain (CRD) and an EPN/WND motif required for carbohydrate-binding specificity. Using tissue expression pattern analysis, OmCTL has been shownto be highly expressed in the liver, and is also detected in other tissues. OmCTL was significantly upregulated in the liver and spleen following infection with Aeromonas hydrophila, suggesting its involvement in immune response. The recombinant OmCTL protein (rOmCTL) agglutinated two gram-negative bacteria, Escherichia coli and A. hydrophila, in vitro in the presence of Ca2+, showing that it is a typical Ca2+-dependent carbohydrate-binding protein.Furthermore, rOmCTL purified from E. coli BL21 (DE3) strongly bound to LPS and PGN, as well as all tested bacteria in a Ca2+-independent manner. These results indicate that OmCTL plays a central role in the innate immune response and as a pattern recognition receptor that recognizes diverse pathogens among O. macrolepis.

[Agglutination and phagocytosis of foreign abiotic particles by bluebottle Calliphora vicina haemocytes in vivo. II. Influence of the previous septic immune induction on haemocytic activity].

The rate of Calliphora vicina haemocytic defense reaction to foreign particles injection depends on the larval age and on the previous bacterial immunization. Immunization of crop-empting larvae induces an evident increase in particles phagocytosis by juvenile plasmatocytes in 24 h after injection. Both the hemogram and the pattern of cellular defense reaction change significantly after crop-empting. Immunized larvae start intensive adhesion of foreign particles to plasmatocytes surface and formation of great aggregations of plasmatocytes (morules) no longer than in 34 min after injection. The period of particle-haemocyte adhesion is short-termed and no more than after 30 min cell aggregates dissociate and adhered charcoal particles pass to thrombocydoidal agglutinates. Unimmunized control larvae of the same age have shown no adhesion and morules formation. In immunized wadering and diapausing larvae, formation of capsules consisting of central thrombocydoidal agglutinate filled with alien particles and adherent plasmatocytes I is intensified. In contrast to moru-les, this capsule formation is not accompanied by charcoal particles adhesion to plasmatocytes. Immunization of mature larvae of C. vicina shown no prominent influence on both the rate of phagocytosis and the hyaline cells differentiation. It might be supposed that the receptors system is complex and the immunization both the mechanisms of foreigners recognition (adhesion, morulation and incapsulation) and the far more lately occurring phagocytosis.

A Clinical Case of Weak A Antigen on the Erythrocytes in a Person with Coexistent Anti-A Antibodies.

This study investigated a person with an AB0 discrepancy. Her blood group initially typed at the birth as AB Rh+ (positive); however, it was B Rh+ (positive) or Rh- (negative) when she was in her teens. At room temperature, her erythrocytes were agglutinated by anti-B, and the agglutination was significantly weaker at 37 ºC. As a result, her erythrocytes did not absorb anti-B but anti-A. Furthermore, her erythrocytes were agglutinated by anti-A at 37 ºC with signs of hemolysis in the presence of complement. The unwashed erythrocytes were also agglutinated in an antiglobulin test by polyclonal anti-A at 37 ºC and by heated polyclonal anti-A and anti-A MAB 2-8 at room temperature. Moreover, her serum agglutinated A erythrocytes at room temperature with less activity at 37 ºC; however, it agglutinated B erythrocytes at 37 ºC. The ability of the erythrocytes of this person to absorb anti-A came along with the agglutination of her erythrocytes at 37 ºC by polyclonal serum and decreased activity of the serum to agglutinate A erythrocytes at 37 ºC, compared to room temperature. The absence of anti-B absorbance by the person’s erythrocytes was accompanied by the presence of anti-B in the serum, which was active at 37 ºC. The incubation of the person’s serum with 0 erythrocytes induced the ability of erythrocytes to absorb anti-A and to be hemolyzed by anti-A in the presence of complement in accordance with the person’s characteristics of erythrocytes. The reaction of absorption and agglutination at room temperature and 37 ºC by heated serum with the use of complement may help to reveal both weak A and B antigens and anti-A and anti-B antibodies while AB0 blood typing.

Sensitive detection of multiple islet autoantibodies in type 1 diabetes using small sample volumes by agglutination-PCR.

Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. However, the reliance on radioactive components, large sample volumes and limited throughput renders radioassay testing costly and challenging. We developed a multiplex analysis platform based on antibody detection by agglutination-PCR (ADAP) for the sample-sparing measurement of GAD, IA-2 and insulin autoantibodies/antibodies in 1 µL serum. The assay was developed and validated in 7 distinct cohorts (n = 858) with the majority of the cohorts blinded prior to analysis. Measurements from the ADAP assay were compared to radioassay to determine correlation, concordance, agreement, clinical sensitivity and specificity. The average overall agreement between ADAP and radioassay was above 91%. The average clinical sensitivity and specificity were 96% and 97%. In the IASP 2018 workshop, ADAP achieved the highest sensitivity of all assays tested at 95% specificity (AS95) rating for GAD and IA-2 autoantibodies and top-tier performance for insulin autoantibodies. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1µL serum with high sensitivity. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies.