Thrombin cleaves recombinant soluble thrombomodulin into a lectin-like domain fragment and a fragment with protein C-activating cofactor activity.

Thrombomodulin (TM) is a transmembrane protein that plays an important role in regulating the coagulation system by acting as a cofactor for thrombin in protein C activation. Additionally, TM is involved in inflammation. Previous studies have shown that soluble fragments of TM of varying sizes, which are derived from membrane-bound TM, are present in plasma and urine. Soluble fragments of TM are speculated to exhibit biological activity. Among these, a lectin-like domain fragment (TMD1) is of particular importance. Recombinant TMD1 has previously been shown to attenuate lipopolysaccharide-induced inflammation. Here, we report that thrombin cleaves recombinant soluble TM, which is used for the treatment of disseminated intravascular coagulation associated with sepsis, into TMD1 and a fragment comprising the C-terminal portion of TM (TMD23), the latter of which retains the cofactor activity for activating protein C. Our findings suggest that thrombin not only activates protein C on membrane-bound TM but may also cleave TM to generate TMD1.

Immunological imprint on peripheral blood in kidney transplant recipients after two doses of SARS-CoV-2 mRNA vaccination in Japan.

The immunological imprint after two doses of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) mRNA vaccination for patients after kidney transplantation (KTx) remain unclear. This study included KTx recipients and volunteer healthy controls (HCs) who received two doses of SARS-CoV-2 mRNA vaccine (Pfizer BioNTech) from January 2021 to December 2021. We analyzed safety within 21 days after each vaccination dose and compared the immune response in peripheral blood mononuclear cells (PBMCs) between the two groups. No graft rejection was observed throughout this study. Adverse events were generally observed within 5 days. The KTx group exhibited a significantly lower degree of symptoms between doses 1 and 2 (P < 0.001). Increases in activated subsets of T and B cells expressing human leukocyte antigen (HLA)-DR and/or CD38 were observed in the HC group after dose 2 (both P < 0.001), with the greatest increases in HLA-DR+CD8+ T cells and CD38+CD19+ B cells (P = 0.042 and P = 0.031, respectively). In addition, PD1+CD8+ T cells-but not PD1+CD4+ T cells-increased significantly in the HC group (P = 0.027). In the KTx group, however, activated HLA-DR+, CD38+, and PD1+ cells remained at baseline levels. Immunoglobulin (Ig)G against SARS-CoV-2 was detected in only four KTx recipients (13.3%) after dose 2 (P < 0.001). Multivariate logistic regression analyses revealed that ΔHLA-DR+CD8+ T cells and ΔCD38+CD19+ B cells were significantly associated with IgG formation (both P = 0.02). SARS-CoV-2 mRNA vaccine generates impaired cellular and humoral immunity for KTx recipients. Results indicate the need for modified vaccination strategies in immunocompromised KTx recipients.

CpG ODN G9.1 as a novel nasal ODN adjuvant elicits complete protection from influenza virus infection without causing inflammatory immune responses.

This study examined the protective efficacy of and immune response to a nasal influenza vaccine combined with a novel mucosal oligodeoxynucleotide (ODN) adjuvant, CpG ODN G9.1 (G9.1), in a model of infection limited to the upper respiratory tract (URT) and a model of infection in the lower respiratory tract (LRT). Mice were nasally primed with an A/California/7/2009 (Cal7) split vaccine (X179A) plus G9.1 and were then nasally given a booster with X179A alone. When mice were challenged with either a large (infection of the LRT) or small (infection limited to the URT) volume of live Cal7 influenza virus, mice nasally given G9.1 combined with X179A had a markedly higher rate of protection against infection limited to the URT. Moreover, this group of mice promptly recovered from an infection of the LRT. When mice were subcutaneously (s.c.) given X179A as a current form of vaccination, they had no protection from an infection limited to the URT but they did recover from an infection of the LRT. The patterns of protection were closely correlated with influenza virus-specific mucosal secretory IgA (SIgA) or serum IgG antibody (Ab) responses. Thus, SIgA Abs responses play an important role in protection from an infection limited to the URT while influenza virus-specific serum IgG Ab responses help to protect from an infection of the LRT. A finding of note is that lungs from mice nasally given G9.1 had low levels of type I IFN-associated protein- and transcription factor-specific mRNA expression. These results suggest that nasal G9.1 can be used as an effective and safe mucosal adjuvant for influenza vaccines since this nasal vaccine system elicits both mucosal SIgA and serum IgG Ab responses that provide complete protection without inducing potent inflammatory responses.

Dual modulating functions of thrombomodulin in the alternative complement pathway.

Thrombomodulin (TM) is a transmembrane protein expressed on vascular endothelial cells. TM has anticoagulant and anti-inflammatory properties. It has recently been reported that TM modulates complement, an immune effector system that destroys pathogens and is also involved in inflammation. TM was demonstrated to enhance the degradation of C3b into iC3b by factor I and factor H, indicating that its role is in negative regulation in the alternative pathway of the complement system. In this study, we examined the effects of recombinant human soluble TM protein composed of the extracellular domains (rTM) on the alternative pathway. The degradation of C3b into iC3b by factor I and factor H was enhanced by rTM as assessed by SDS-PAGE, confirming the previous observation. We also found that rTM enhances the cleavage of C3 into C3b as a result of activation of the alternative pathway. These results indicate that TM has both activating and inactivating functions in the alternative pathway.

CTRP6 is an endogenous complement regulator that can effectively treat induced arthritis.

The complement system is important for the host defence against infection as well as for the development of inflammatory diseases. Here we show that C1q/TNF-related protein 6 (CTRP6; gene symbol C1qtnf6) expression is elevated in mouse rheumatoid arthritis (RA) models. C1qtnf6(-/-) mice are highly susceptible to induced arthritis due to enhanced complement activation, whereas C1qtnf6-transgenic mice are refractory. The Arthus reaction and the development of experimental autoimmune encephalomyelitis are also enhanced in C1qtnf6(-/-) mice and C1qtnf6(-/-) embryos are semi-lethal. We find that CTRP6 specifically suppresses the alternative pathway of the complement system by competing with factor B for C3(H2O) binding. Furthermore, treatment of arthritis-induced mice with intra-articular injection of recombinant human CTRP6 cures the arthritis. CTRP6 is expressed in human synoviocytes, and CTRP6 levels are increased in RA patients. These results indicate that CTRP6 is an endogenous complement regulator and could be used for the treatment of complement-mediated diseases.

Purification, measurement of concentration, and functional complement assay of human ficolins.

Ficolins constitute a group of lectins involved in innate immunity. L-Ficolin, H-ficolin, and M-ficolin are present in human serum. The human ficolins differ in carbohydrate-binding specificity, but they have in common the ability to recognize the acetyl group. L-Ficolin and H-ficolin are associated with serine proteases termed MASPs (MBL-associated serine proteases) and their truncated proteins, and the complexes (L/H-ficolin-MASP) activate the lectin pathway of complement upon binding to their ligands. Recombinant M-ficolin is also able to form a complex with MASP, resulting in complement activation. L-Ficolin and H-ficolin can be purified as a complex with MASP from serum by utilizing their binding specificities. These ficolin-MASP complexes have an ability to activate C4. Human ficolins are quantified by ELISA using specific antibodies or ligands.

Glycoepitopes of staphylococcal wall teichoic acid govern complement-mediated opsonophagocytosis via human serum antibody and mannose-binding lectin.

Serum antibodies and mannose-binding lectin (MBL) are important host defense factors for host adaptive and innate immunity, respectively. Antibodies and MBL also initiate the classical and lectin complement pathways, respectively, leading to opsonophagocytosis. We have shown previously that Staphylococcus aureus wall teichoic acid (WTA), a cell wall glycopolymer consisting of ribitol phosphate substituted with α- or ß-O-N-acetyl-d-glucosamine (GlcNAc) and d-alanine, is recognized by MBL and serum anti-WTA IgG. However, the exact antigenic determinants to which anti-WTA antibodies or MBL bind have not been determined. To answer this question, several S. aureus mutants, such as α-GlcNAc glycosyltransferase-deficient S. aureus ΔtarM, ß-GlcNAc glycosyltransferase-deficient ΔtarS, and ΔtarMS double mutant cells, were prepared from a laboratory and a community-associated methicillin-resistant S. aureus strain. Here, we describe the unexpected finding that ß-GlcNAc WTA-deficient ΔtarS mutant cells (which have intact α-GlcNAc) escape from anti-WTA antibody-mediated opsonophagocytosis, whereas α-GlcNAc WTA-deficient ΔtarM mutant cells (which have intact ß-GlcNAc) are efficiently engulfed by human leukocytes via anti-WTA IgG. Likewise, MBL binding in S. aureus cells was lost in the ΔtarMS double mutant but not in either single mutant. When we determined the serum concentrations of the anti-α- or anti-ß-GlcNAc-specific WTA IgGs, anti-ß-GlcNAc WTA-IgG was dominant in pooled human IgG fractions and in the intact sera of healthy adults and infants. These data demonstrate the importance of the WTA sugar conformation for human innate and adaptive immunity against S. aureus infection.

Activation of the alternative complement pathway by mannose-binding lectin via a C2-bypass pathway.

MBL is a serum lectin that activates the lectin pathway of the complement system. MBL forms complexes with three types of MASPs. Upon binding to Salmonella serogroup C-specific oligosaccharide, MBL activates the alternative pathway via a C2-bypass pathway without involving MASP-2, C2 or C4. We demonstrate that mannan-bound MBL activates the alternative pathway via a C2-bypass pathway that requires MASP-2 and C4. Thus, depending on the ligands to which MBL binds, there may be two distinct MBL-mediated C2-bypass pathways.

Characterization of the complex between mannose-binding lectin trimer and mannose-binding lectin-associated serine proteases.

Mannose-binding lectin (MBL) is an oligomeric serum lectin involved in innate immunity. Human MBL is complexed with three types of serine proteases (MASP-1, MASP-2 and MASP-3) and two types of their truncated forms (sMAP and MAp44). When an MBL complex binds to carbohydrates of pathogens, the complement system is activated via the lectin pathway. Human MBL is a mixture of different sized oligomers that range mainly from trimers to hexamers. It has been suggested that different MBL oligomers may have distinct MASP compositions. In the present study, an MBL trimer (MBL-I) exclusive of other oligomers was isolated from human serum by chromatography. Immunoblot analysis of MBL-I revealed that it had been co-purified with MASP-1 and sMAP. This suggests that MASP-1 and sMAP are bound to each other in MBL-I. The MBL-I complex was found to activate C2, but to lack the ability to activate C4 due to the absence of MASP-2.

Human serum mannose-binding lectin senses wall teichoic acid Glycopolymer of Staphylococcus aureus, which is restricted in infancy.

Innate immunity is the first line of host defense against invading pathogens, and it is recognized by a variety of pattern recognition molecules, including mannose-binding lectin (MBL). MBL binds to mannose and N-acetylglucosamine residues present on the glycopolymers of microorganisms. Human serum MBL functions as an opsonin and activates the lectin complement pathway. However, which glycopolymer of microorganism is recognized by MBL is still uncertain. Here, we show that wall teichoic acid of Staphylococcus aureus, a bacterial cell surface glycopolymer containing N-acetylglucosamine residue, is a functional ligand of MBL. Whereas serum MBL in adults did not bind to wall teichoic acid because of an inhibitory effect of anti-wall teichoic acid antibodies, MBL in infants who had not yet fully developed their adaptive immunity could bind to S. aureus wall teichoic acid and then induced complement C4 deposition. Our data explain the molecular reasons of why MBL-deficient infants are susceptible to S. aureus infection.

An alternative method for vermilion reconstruction after resection of hemangiomas of the lip.

PURPOSE: We describe a method for symmetrical vermilion reconstruction after resection of hemangiomas of the lip. PATIENTS AND METHODS: Four patients underwent vermilion reconstruction after resection of large cavernous hemangiomas of the lip. This reconstruction technique employed 3 basic components: 1 ) labial mucosal advancement flap, 2 ) orbicularis oris muscle flap, if necessary, and 3 ) free mucosal graft. RESULTS: All patients successfully underwent the planned procedures without significant complications. Symmetrical profiles of vermilion of the lip were achieved in all cases, even when an extended excision had been performed. CONCLUSIONS: We have found that the combination of labial mucosal advancement flaps, with or without muscular flaps, and free mucosal grafts provides excellent esthetic outcomes with a low complication rate. This method should be incorporated into the surgical techniques for symmetrical reconstruction after resection of hemangiomas of the lip.