Kazuko Kanno

Mannose-Binding Lectin (MBL)-Associated Serine Protease (MASP)-1 Contributes to Activation of the Lectin Complement Pathway

The complement system plays an important role in innate immunity. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme. It has been suggested that MASP-2 is responsible for the activation of C4. Other serine proteases (MASP-1 and MASP-3) are also associated with MBL or ficolins; however, their functions are still controversial. In this study, a MASP-1- and MASP-3-deficient mouse model (MASP1/3−/−) was generated by a gene targeting strategy to investigate the roles of MASP-1 and MASP-3 in the lectin pathway. Serum derived from MASP1/3−/− mice showed significantly lower activity of both C4 and C3 deposition on mannan-agarose, and this low activity was restored by the addition of recombinant MASP-1. MASP-1/3-deficient serum showed a significant delay for activation of MASP-2 compared with normal serum. Reconstitution of recombinant MASP-1 in MASP-1/3-deficient serum was able to promote the activation of MASP-2. From these results, we propose that MASP-1 contributes to the activation of the lectin pathway, probably through the activation of MASP-2.

Essential role of Mannose-binding lectin-associated serine protease-1 in activation of the complement factor D

The complement system is an essential component of innate immunity, participating in the pathogenesis of inflammatory diseases and in host defense. In the lectin complement pathway, mannose-binding lectin (MBL) and ficolins act as recognition molecules, and MBL-associated serine protease (MASP) is a key enzyme; MASP-2 is responsible for the lectin pathway activation. The function of other serine proteases (MASP-1 and MASP-3) is still obscure. In this study, we generated a MASP-1– and MASP-3–deficient mouse model (Masp1/3−/−) and found that no activation of the alternative pathway was observed in Masp1/3−/− serum. Mass spectrometric analysis revealed that circulating complement factor D (Df) in Masp1/3−/− mice is a zymogen (pro-Df) with the activation peptide QPRGR at its N terminus. These results suggested that Masp1/3−/− mice failed to convert pro-Df to its active form, whereas it was generally accepted that the activation peptide of pro-Df is removed during its secretion and factor D constitutively exists in an active form in the circulation. Furthermore, recombinant MASP-1 converted pro-Df to the active form in vitro, although the activation mechanism of pro-Df by MASP-1 is still unclear. Thus, it is clear that MASP-1 is an essential protease of both the lectin and alternative complement pathways.

The Role of Mannose-Binding Lectin-Associated Serine Protease-3 in Activation of the Alternative Complement Pathway

Mannose-binding lectin (MBL)-associated serine proteases (MASPs) are responsible for activation of the lectin complement pathway. Three types of MASPs (MASP-1, MASP-2, and MASP-3) are complexed with MBL and ficolins in serum. Although MASP-1 and MASP-2 are known to contribute to complement activation, the function of MASP-3 remains unclear. In this study, we investigated the mechanism of MASP-3 activation and its substrate using the recombinant mouse MASP-3 (rMASP-3) and several different types of MASP-deficient mice. A proenzyme rMASP-3 was obtained that was not autoactivated during preparation. The recombinant enzyme was activated by incubation with Staphylococcus aureus in the presence of MBL-A, but not MBL-C. In vivo studies revealed the phagocytic activities of MASP-1/3–deficient mice and all MASPs (MASP-1/2/3)–deficient mice against S. aureus and bacterial clearance in these mice were lower than those in wild-type and MASP-2–deficient mice. Sera from all MASPs-deficient mice showed significantly lower C3 deposition activity on the bacteria compared with that of wild-type serum, and addition of rMASP-3 to the deficient serum restored C3 deposition. The low C3 deposition in sera from all MASPs-deficient mice was probably caused by the low level factor B activation that was ameliorated by the addition of rMASP-3. Furthermore, rMASP-3 directly activated factors B and D in vitro. These results suggested that MASP-3 complexed with MBL is converted to an active form by incubation with bacterial targets, and that activated MASP-3 triggered the initial activation step of the alternative complement pathway.

Small Mannose-Binding Lectin-Associated Protein Plays a Regulatory Role in the Lectin Complement Pathway

Mannose-binding lectin (MBL) and ficolins are pattern recognition proteins acting in innate immunity, and they trigger the activation of the lectin complement pathway through MBL-associated serine proteases (MASPs). Upon activation of the lectin pathway, MASP-2 cleaves C4 and C2. A truncated form of MASP-2, named small MBL-associated protein (sMAP), is also associated with MBL/ficolin-MASP complexes. To clarify the role of sMAP, we have generated sMAP-deficient (sMAP−/−) mice by targeted disruption of the sMAP-specific exon. Because of the gene disruption, the expression level of MASP-2 was also decreased in sMAP−/− mice. When recombinant sMAP (rsMAP) and recombinant MASP-2 (rMASP-2) reconstituted the MBL-MASP-sMAP complex in deficient serum, the binding of these recombinant proteins to MBL was competitive, and the C4 cleavage activity of the MBL-MASP-sMAP complex was restored by the addition of rMASP-2, whereas the addition of rsMAP attenuated the activity. Therefore, MASP-2 is essential for the activation of C4 and sMAP plays a regulatory role in the activation of the lectin pathway.

Mice Deficient in Ficolin, a Lectin Complement Pathway Recognition Molecule, Are Susceptible to Streptococcus pneumoniae Infection

Mannose-binding lectin (MBL) and ficolin are complexed with MBL-associated serine proteases, key enzymes of complement activation via the lectin pathway, and act as soluble pattern recognition molecules in the innate immune system. Although numerous reports have revealed the importance of MBL in infectious diseases and autoimmune disorders, the role of ficolin is still unclear. To define the specific role of ficolin in vivo, we generated model mice deficient in ficolins. The ficolin A (FcnA)–deficient (Fcna−/−) and FcnA/ficolin B double-deficient (Fcna−/−b−/−) mice lacked FcnA-mediated complement activation in the sera, because of the absence of complexes comprising FcnA and MBL-associated serine proteases. When the host defense was evaluated by transnasal infection with a Streptococcus pneumoniae strain, which was recognized by ficolins, but not by MBLs, the survival rate was significantly reduced in all three ficolin-deficient (Fcna−/−, Fcnb−/−, and Fcna−/−b−/−) mice compared with wild-type mice. Reconstitution of the FcnA-mediated lectin pathway in vivo improved survival rate in Fcna−/− but not in Fcna−/−b−/− mice, suggesting that both FcnA and ficolin B are essential in defense against S. pneumoniae. These results suggest that ficolins play a crucial role in innate immunity against pneumococcal infection through the lectin complement pathway.

Unique Phenotypes of C1s Deficiency and Abnormality Caused by Two Compound Heterozygosities in a Japanese Family

A deficiency in the early components of complement is associated with an increased susceptibility to pyrogenic infections and multiple autoimmune diseases. We previously reported a Japanese case of selective C1s deficiency resulting from a compound heterozygosity for a 4-bp deletion in exon X and a nonsense mutation Glu597X in exon XII of the C1s gene. In this previous case, the patient suffered from unique symptoms including virus-associated hemophagocytic syndrome and died after a long period of loss of consciousness. In the present study, we report another patient from the same family, with C1s abnormality caused by a distinct compound-heterozygous genotype and who had a novel missense mutation Gly630Glu transmitted from the mother’s side and a previously identified nonsense mutation Glu597X from the father’s side. Thus three distinct mutations of the C1s gene were clustered and resulted in two distinct genotypes for C1s deficiency and C1s abnormality within this one family. The present patient showed symptoms that were similar in part to our previous patient, which were different from those of the cases reported in other families. The biochemical properties of C1s in the patient’s serum and the recombinant form were closely related to the undetectable or very low activity of complement activation. These results suggested that the uniqueness and severity of the symptoms observed here in the two patients might be under the control of a common C1s allele and distinct counterparts, respectively.

Role of complement in a murine model of peanut-induced anaphylaxis.

Peanut allergy is severe and persisting from childhood to adulthood. However, there is no effective prophylaxis or treatment for peanut allergy. Little is known to about the molecular process in the pathogenesis of peanuts allergy, especially in innate immunity. Thus we investigated the role of complement activation in murine peanut anaphylaxis. Complement component C3 deposition on peanut extract (PE) was evaluated using sera from wild-type (WT), mannose-binding lectin associated serine protease (MASP)-1/3 deficient, MASP-2 deficient, and C4 deficient mice. Sera from interferon regulatory factor-4 (IRF-4) deficient mice, which lack serum immunoglobulin, were also used. In anaphylaxis study, mice were pretreated with propranolol and a long-acting form of IL-4, and injected with PE. Mice were then assessed for plasma C3a levels and hypothermia shock by ELISA and rectal temperature measurement, respectively. C3 deposition on PE was abolished in immunoglobulin- and C4-deficient sera. No difference in C3 deposition levels were observed among WT, MASP-1/3 deficient and MASP-2 deficient sera. IgM, IgG2b, IgG3, C1q, and ficolin-A deposits were detected on PE. In anaphylaxis study, MASP-1/3 deficient mice showed elevation of plasma C3a levels similar to WT mice. However, they were significantly reduced in C4- and MASP-2-deficient mice compared to WT mice. Consistently, PE-induced anaphylactic shock was prevented in C4 deficient mice and partially in MASP-2 deficient mice. In conclusion, PE activates complement via both the lectin and classical pathways in vivo, and the complement activation contributes to hypothermia shock in mice.

The Relationship between Mismatch Negativity and the COMT Val108/158Met Genotype in Schizophrenia

Mismatch negativity (MMN) is a component of auditory event-related potentials that reflects automatic change detection in the brain, showing qualities of endophenotypes in schizophrenia. MMN deficiency is one of the robust findings in patients, and it reflects both cognitive and functional decline. Catechol-o-methyltransferase (COMT) is a key enzyme involved in regulating dopamine transmission within the prefrontal cortex. A preliminary study suggested that the COMTVal108/158Met genotype (rs4680) is related to cognitive function in schizophrenia. Both the COMTVal108/158Met genotype and MMN are related to cognitive function, but no studies have reported on the relationship between MMN and the COMTVal108/158Met genotype in schizophrenia. This study therefore examined the relationship between COMTVal108/158Met genotype and MMN. The duration of MMN was measured, and the COMTVal108/158Met polymorphism was detected by polymerase chain reaction-restriction fragment length polymorphism in 49 Japanese schizophrenia patients (Val/Val, n = 21; Met carriers, n = 28). Amplitude and latency of MMN were compared between Val/Val and Met carriers.

PM446. The relationship between MMN and COMT Val108/158Met genotype in schizophrenia

Cognitive deficits (CD) in schizophrenia are recalcitrant to treatment as usual. Whilst there has been considerable interest in recent years for evaluating the efficacy of cognitive remediation (CR) programs in schizophrenia at research settings, scant attention has been paid to evaluate the effectiveness of CR programs at everyday clinical practice settings. Method: We evaluated retrospectively short-term cognitive, occupational and accommodation outcomes of consecutive patients with schizophrenia admitted over a 5 year period at a tertiary-care inpatient public psychiatric rehabilitation facility in Western Australia. The Brief Assessment of Cognition in Schizophrenia (BACS) was utilised to assess cognition. Patients were divided into 3 groups based on their participation in the neuroplasticity based auditory CR program of PositScience; those who did not participate (non-trainers), those who completed less than 20 hours of training (incomplete-trainers) and those who completed 20 or more hours of training (completetrainers). Results: The mean age of the patients was 32.1 years, 68.5% were males, nearly 80% had treatment-resistant illness, 65% were on clozapine and comorbidity was highly prevalent (72%). Thirtyseven patients were classified as non-trainers, 17 as incompletetrainers and 34 as completetrainers. The 3 groups did not differ in measured demographic and clinical parameters. Compared to their admission scores, completetrainers had significantly improved scores at discharge on verbal memory (p=0.012), motor speed (p=0.009) and the composite score (p=0.006). Furthermore, 24%, 22% and 36% of patients changed from unemployed to the employed group in the non-trainers, incomplete-trainers and complete-trainers groups respectively from admission to discharge. Conclusion: Our study demonstrates that CR program can be integrated effectively into the interventions provided for people with schizophrenia at public psychiatric rehabilitation settings. Significant improvements in cognitive and functional outcomes revealed in this study indicate the need for further translational research in the field of CR in schizophrenia. PM445 A family of primary familial brain calcification due to mutation in platelet-derived growth factor-B gene Teruo Hayashi, Giovanni Coppola, Andrea Legati, Tadashi Nishikawa Seiwakai Nishikawa Hospital, Japan Abstract Primary familial brain calcification (PFBC) is a neuropsychiatric disorder characterized by abnormal deposits of calcium in the basal ganglia and cerebellum. PFBC can present with a spectrum of symptoms resembling those seen in dementia and schizophrenia. Mutations in some genes have been found to cause PFBC: namely, the SLC20A2 gene that codes for the sodium-dependent phosphate transporter and the PDGFRB gene that codes for the platelet-derived growth factor (PDGF) receptor β. A recent study found that mutations of PDGFB, which encode the ligand peptide PDGF-B for the PDGF receptor β, also cause PFBC. Here we report the first Japanese family of PFBC carrying a mutation of PDGFB. CT scans revealed a symmetrical calcification over the basal ganglia in two members of the family. One family member complained auditory hallucination at 16 years old, and had been treated for schizophrenia. The other family member complained memory and gait disturbances in his late 60s. The mutation in PDGFB (c.445C>T, p.Arg149*) that causes the substitution of an arginine with a stop codon at amino acid 149 of the PDGF-B protein (p. Arg149*) was consistently detected in both cases. No mutations in SLC20A2 were detected. This finding indicates that the PDGF pathway plays a crucial role in pathogenesis of PFBC, and that dysfunction of the PDGF signaling may lead to psychiatric symptoms that are associated with dementia and/or schizophrenia.Primary familial brain calcification (PFBC) is a neuropsychiatric disorder characterized by abnormal deposits of calcium in the basal ganglia and cerebellum. PFBC can present with a spectrum of symptoms resembling those seen in dementia and schizophrenia. Mutations in some genes have been found to cause PFBC: namely, the SLC20A2 gene that codes for the sodium-dependent phosphate transporter and the PDGFRB gene that codes for the platelet-derived growth factor (PDGF) receptor β. A recent study found that mutations of PDGFB, which encode the ligand peptide PDGF-B for the PDGF receptor β, also cause PFBC. Here we report the first Japanese family of PFBC carrying a mutation of PDGFB. CT scans revealed a symmetrical calcification over the basal ganglia in two members of the family. One family member complained auditory hallucination at 16 years old, and had been treated for schizophrenia. The other family member complained memory and gait disturbances in his late 60s. The mutation in PDGFB (c.445C>T, p.Arg149*) that causes the substitution of an arginine with a stop codon at amino acid 149 of the PDGF-B protein (p. Arg149*) was consistently detected in both cases. No mutations in SLC20A2 were detected. This finding indicates that the PDGF pathway plays a crucial role in pathogenesis of PFBC, and that dysfunction of the PDGF signaling may lead to psychiatric symptoms that are associated with dementia and/or schizophrenia. PM446 The relationship between MMN and COMT Val108/158Met genotype in schizophrenia Sho Horikoshi,Tetsuya Shiga, Hiroshi Hoshino, Haruka Ochiai, Keiko Kanno-Nozaki, Kazuko Kanno, Haruka Kaneko, Itaru Miura, Hirooki