Serial change of C1 inhibitor in patients with sepsis--a preliminary report.

OBJECTIVE: C1 inhibitor (C1INH) regulates not only the complement system but also the plasma kallikrein-kinin, fibrinolytic, and coagulation systems. The biologic activities of C1INH can be divided into the regulation of vascular permeability and anti-inflammatory functions. The objective was to clarify the serial change of C1INH in patients with sepsis. METHODS: We serially examined C1INH activity values (reference range, 70%-130%) and quantitative values (reference range, 160-330 µg/mL) in patients with sepsis admitted into the intensive care unit of the Trauma and Acute Critical Care Center at Osaka University Hospital (Osaka, Japan) during the period between December 2012 and February 2013. We also analyzed their clinical course. We defined "refractory shock" as septic shock requiring steroid administration to maintain hemodynamics. RESULTS: The serial change of C1INH was evaluated in 5 patients (4 survivors and 1 nonsurvivor). Two patients were diagnosed as having refractory shock. In the nonsurvivor after refractory shock, C1INH activity on admission was 97.2%, and the quantitative value was 133.1 µg/mL. In the other patient with refractory shock, C1INH activity on admission was 94.4%, and the quantitative value was 126.7 µg/mL. This patient's general condition had improved by day 6, with increases in C1INH activity (139.9%) and quantitative value (250.1 µg/mL). In the 3 nonrefractory shock patients, C1INH activity on admission was 130.6%±8.7%, and the quantitative value was 215±26.5 µg/mL. CONCLUSIONS: Enhancement of C1INH activity was not observed in the refractory shock patients, and the C1INH quantitative values were low. Further evaluation of the serial change of C1INH and the validity of C1INH replacement therapy in patients with septic shock may lead to a new strategy for sepsis management.

Thomsen-Friedenreich antigen exposure as a cause of Streptococcus pyogenes-associated hemolytic-uremic syndrome.

Infection with Streptococcus pyogenes, a Group A beta-hemolytic streptococcus (GAS), is a rare cause of hemolyticuremic syndrome (HUS). Invasive infections with Streptococcus pneumoniae that produce neuraminidase are a well-recognized cause of HUS without diarrhea. The Thomsen- Friedenreich antigen (T antigen) plays a role in the pathophysiology of pneumococcal HUS. We describe the case of a 3-year-old boy with GAS-associated HUS and show how T-antigen exposure was implicated in this case. He had no diarrhea and cultures for blood, urine, and stool were negative. The urinary pneumococcal antigen was negative; his direct Coombs test was positive. Glomerular capillary loops, tubular epithelium on his renal biopsy specimen, and red blood cells in his blood smear showed positive fluorescence with anti-T lectin. Although the pathogenesis of GAS-associated HUS is not well understood, T-antigen exposure may be implicated in some cases with GAS-associated HUS.

Successful pregnancy in a patient suffering from recurrent mid-trimester miscarriage with C9 deficiency after receiving cervical cerclage followed by clindamycin and progesterone: a case report.

Complement component 9 (C9) deficiency is relatively common, especially in Japan. Here we present the case of a 27-year-old Japanese woman whose obstetric history involved three mid-trimester miscarriages (at 22 weeks', 18 weeks' and 21 weeks' gestation) and one early spontaneous miscarriage. Her fifth pregnancy was successfully managed by cervical cerclage at 13 weeks' gestation, followed by clindamycin administration (600 mg/day for 7 days) and progesterone injections (250 mg/week). She gave birth to a healthy 3326-g male infant at 40 weeks and 1 day gestation after natural onset of labor. After delivery, the serum complement components were analyzed. C9 protein and activity were undetectable in the patient's serum. We suggest that an immunologic disorder such as C9 deficiency should be considered as a potential complication of undiagnosed recurrent miscarriages.

Etanercept is safely used for treating psoriatic arthritis in a patient complicated with type 1 hereditary angioedema.

Hereditary angioedema (HAE) is a life-threatening disorder caused by deficiency or dysfunction of the C1 inhibitor protein. Patients with HAE are restricted in various medical treatments, which can induce an HAE attack. We herein report the first case of psoriatic arthritis (PSA) with type 1 HAE successfully treated with 25 mg of etanercept without HAE attack. Etanercept may represent a useful choice for treating patients with HAE accompanied by intractable PSA and rheumatoid arthritis (RA).

A case of dense deposit disease associated with a group A streptococcal infection without the involvement of C3NeF or complement factor H deficiency.

A 14-year-old girl presented with acute glomerulonephritis. Tests revealed hypocomplementemia and elevated Antistreptolysin-O titers, and renal biopsy revealed endocapillary and mesangial proliferative glomerulonephritis with double contours of the glomerular basement membrane (GBM). Despite methylprednisolone pulse therapy and the administration of oral prednisolone, overt proteinuria and hypocomplementemia persisted. A second renal biopsy 6 months later confirmed the initial diagnosis of dense deposit disease (DDD) based on electron-dense deposits in the GBM. C3 nephritic factor (C3NeF) and a deficiency of complement factor H (CFH) were not evident. A nephritis-associated plasmin receptor (NAPlr), nephritogenic group A streptococcal antigen, and the plasmin activity by in situ zymography were been in both the first and second biopsy specimens. The patient received combined immunomodulatory therapy with prednisolone and mizoribine, and the urinary protein decreased to a mild level at 27 months after disease onset. These findings suggest that persistent glomerular NAPlr deposition may be associated with the pathogenesis of DDD in some patients without the involvement of C3NeF or CFH mutation and that DDD patients of this type may respond to immunomodulatory treatment.

Studies of monkey complement: measurement of cynomolgus monkey CH50, ACH50, C4, C2 and C3.

BACKGROUND: The cynomolgus monkey is commonly used as the recipient in transplantation experiments. However, study of the complement system of cynomolgus monkeys is lacking. In the present study, the complement system of cynomolgus monkeys was compared with that of humans, by checking hemolytic titers. METHODS: Hemolytic titers of complement from cynomolgus monkeys were calculated using the same methods as are used in humans. The complement regulatory function of human decay accelerating factor (DAF, CD55) in cynomolgus monkey serum was next studied using erythrocytes from human DAF-transgenic pigs. RESULTS: The results indicated relatively high values, except for C4: CH50: 211.19 +/- 42.78 units/ml, ACH50: 51.47 +/- 12.43 units/ml, C4: 30 170 +/- 14 300 SFU/ml C2: 33831 +/- 7442 SFU/ml and C3: 93612 +/- 30131 SFU/ml. Western blot experiments using antibodies for human complement components revealed similarities between the cynomolgus monkey and human complement systems. Human DAF inhibited pig erythrocyte lysis from approximately 60-70% to 17% in both human and cynomolgus monkey sera, indicating an almost identical complement regulatory function. CONCLUSION: The hemolytic titer of cynomolgus monkeys was greater than the titer measured in human serum. However, human DAF showed nearly the same complement regulatory function in both human and cynomolgus monkey sera.

Alternative pathway activation due to low level of complement factor H in primary antiphospholipid syndrome.

INTRODUCTION: Although complement activation has been proposed as a possible thrombophilic mechanism in antiphospholipid syndrome (APS), the origin of complement activation in APS remains unclear. Here, we focused on complement regulatory factors (CRF), which control the complement system to prevent damage to host tissue. We evaluated the function of two major CRF, membrane cofactor protein (MCP) and factor H (FH), in APS patients. MATERIALS AND METHODS: In this study, we analyzed preserved serum samples from 27 patients with primary APS (PAPS), 20 with APS complicated with SLE (APS + SLE), 24 with SLE (SLE), and 25 with other connective tissue diseases (Other CTD). Serum MCP and FH levels were tested by ELISA. Autoantibodies against FH were determined by both ELISA and western-blotting. RESULTS: Serum complement levels of PAPS were lower than those of other CTD (median C3: 82 vs 112 mg/dL, p < 0.01, C4: 15 vs 22 mg/dL, p < 0.05). Serum MCP levels did not significantly differ among the groups. Serum FH levels were significantly lower in PAPS patients compared with SLE or other CTD (median 204, 1275, and 1220 µg/mL, respectively, p < 0.01). In PAPS patients, serum FH levels were positively correlated with serum C3 levels (p < 0.01, R = 0.55), but no correlation was found with serum C4 levels (p = 0.22, R = 0.33). Autoantibodies against FH were not detected in any of our patients. CONCLUSIONS: Activation of the alternative complement pathway due to low level of FH is one of the possible thrombophilic mechanisms in PAPS.

Studies of innate immune systems against human cells.

BACKGROUND: Pigs are frequently used as animal models for experiments in the surgical field, including allo- and xeno-transplantation. Regeneration studies, including studies dealing with human- and monkey-induced pluripotent stem cells (iPSC), have gradually progressed, with pigs sometimes being used as the scaffold. However, the immunological response of pigs against humans, especially innate immunities, remain unclear. This study reports on a comprehensive study of pig innate immunity against humans. METHODS: Hemolytic complement activity of pig serum was measured using a microtitration technique. The pig natural anti-human antibody (Ab) was examined using human peripheral blood mononuclear cells (PBMC). The reaction of pig natural killer (NK) cells and monocytes/macrophages against human cells was also assessed. RESULTS: Most of the pig complement titers were measured based on methods used in human complement assays. The alternative pathway for pig complement reacts with human cells, indicating that pig complement can react with human cells. Pig serum contains relatively high levels of natural antibodies, IgM and IgG, to human PBMC. Furthermore, the killing of NK cells- and monocyte/macrophage-mediated human cells was clearly confirmed. CONCLUSION: The collective findings indicate that the pig innate immunological systems, not only serum but also cellular factors, are able to recognize and injure human cells.

A case of fibrillary glomerulonephritis associated with thrombotic microangiopathy and anti-glomerular basement membrane antibody.

We present the first report of a case of fibrillary glomerulonephritis (FGN) associated with thrombotic microangiopathy (TMA) and anti-glomerular basement membrane antibody (anti-GBM antibody). A 54-year-old man was admitted to our hospital for high fever and anuria. On the first hospital day, we initiated hemodialysis for renal dysfunction. Laboratory data revealed normocytic-normochromic anemia with schistocytes in the peripheral smear, thrombocytopenia, increased serum lactate dehydrogenase, decreased serum haptoglobin, and negative results for both direct and indirect Coombs tests. Based on these results, we diagnosed TMA. Assays conducted several days later indicated a disintegrin-like and metalloprotease with a thrombospondin motif 13 (ADAMTS13) activity of 31.6%, and ADAMTS13 inhibitors were negative. We started plasma exchange using fresh frozen plasma and steroid pulse therapy. Anti-GBM antibody was found to be positive. Renal biopsy showed FGN. Blood pressure rose on the 46th hospital day, and mild convulsions developed. Based on magnetic resonance imaging of the head, the patient was diagnosed with reversible posterior leukoencephalopathy syndrome. Hypertension persisted despite administration of multiple antihypertensive agents, and the patient experienced a sudden generalized seizure. Computed tomography of the head showed multiple cerebral hemorrhages. However, his blood pressure subsequently decreased and the platelet count increased. TMA remitted following 36 plasma exchange sessions, but renal function was not restored, and maintenance hemodialysis was continued. The patient was discharged on the 119th day of hospitalization. In conclusion, it was shown that TMA, FGN and anti-GBM antibody were closely related.

Development of a novel cytomedical treatment that can protect entrapped cells from host humoral immunity.

Cell therapy is expected to relieve the shortage of donors needed for organ transplantation. When patients are treated with allogeneic or xenogeneic cells, it is necessary to develop a means by which to isolate administered cells from an immune attack by the host. We have developed "cytomedicine," which consists of functional cells entrapped in semipermeable polymer, and previously reported that alginate-poly-L-lysine-alginate microcapsules and agarose microbeads could protect the entrapped cells from injury by cellular immunity. However, their ability to isolate from humoral immunity was insufficient. It is well known that the complement system plays an essential role in rejection of transplanted cells by host humoral immunity. Therefore, the goal of the present study was to develop a novel cytomedical device containing a polymer capable of inactivating complement. In the screening of various polymers, polyvinyl sulfate (PVS) exhibited high anticomplement activity and low cytotoxicity. Murine pancreatic beta-cell line (MIN6 cell) entrapped in agarose microbeads containing PVS maintained viability and physiological insulin secretion, replying in response to glucose concentration, and resisted rabbit antisera in vitro. PVS inhibited hemolysis of sensitized sheep erythrocytes (EAs) and rabbit erythrocytes by the complement system. This result suggests that PVS inhibits both the classical and alternative complement pathways of the complement system. Next, the manner in which PVS exerts its effects on complement components was examined. PVS was found to inhibit generation of C4a and Ba generation in activation of the classical and alternative pathways, respectively. Moreover, when the EAC1 cells, which were carrying C1 on the EAs, treated with PVS were exposed to C1-deficient serum, hemolysis decreased in a PVS dose-dependent manner. These results suggest that PVS inhibits C1 in the classical pathway and C3 convertase formation in the alternative pathway. Therefore, PVS may be a useful polymer for developing an anticomplement device for cytomedical therapy.

Synthesis of factor D by gastric cancer-derived cell lines.

Synthesis of complement components in vitro by four human gastric cancer-derived cell lines, MKN28, MKN74, MKN45 and KATO-III, was studied. When these cells were cultured for 3 days without addition of any stimulator, 0.94 +/- 0.49, 2.10 +/- 0.59, 7.29 +/- 5.94 and 2.47+/- 1.34 ng of factor D/10(6) cells were detected in supernatants of MKN28, MKN74, MKN45 and KATO-III, respectively. Factor D production by these cells was reversibly inhibited by the presence of cycloheximide. Factors B, C3 and C2 were also detected in protein-free culture medium of these cell lines. Addition of tumour necrosis factor (TNF) to culture enhanced C3 and factor B secretion but depressed C2 secretion, without any distinct effect on factor D secretion. Since all cell lines tested secreted significant amounts of factor D without addition of any stimulator in medium, it is possible that factor D may be synthesized by gastric epithelial cells physiologically and constitutively. From a quantitative analysis of factor D secretion by these cells, factor D secreted by gastric tissue is likely to contribute to the factor D level in circulating blood. The possible mechanism of participation of complement system in inflammation of gastric epithelium was proposed. Thus, the present study may be significant for clarification of the mode of extrahepatic complement synthesis participating in mucosal immunity.

Deposition of complement protein C3b on mixed self-assembled monolayers carrying surface hydroxyl and methyl groups studied by surface plasmon resonance.

Since complement activation is recognized as a common response of the host defense system when an artificial medical device is applied to a patient, great effort has been devoted to studies on the interaction of the complement system with artificial materials. However, some uncertainties remain, partially because of the lack of well characterized surfaces and suitable analytic methods for study of the surface phenomena that occur on artificial materials under physiologic conditions. In this study, we employed self-assembled monolayers (SAMs) and the surface plasmon resonance (SPR) technique to study interactions of the serum complement with well characterized surfaces. Self-assembled monolayers carrying various concentrations of hydroxyl groups were prepared using 11-mercapto-1-undecanol (C11-OH) and one of n-nonanethiol, n-dodecanethiol, and n-hexadecanethiol. The amount of NHS deposition on the SAMs increased with increasing C11-OH content of the SAMs, and the amount of anti-C3b antibody immobilization formed on the NHS deposition layers increased with increasing C11-OH content of the SAMs. These results clearly demonstrate that a large amount of C3b, produced through the activation of the complement system, binds covalently to and is adsorbed by hydroxyl-group-rich surfaces. The combination of SAMs and the SPR technique is suitable for studying the interaction of the complement system with solid surfaces, and the results should give basic information needed for a rational design of biocompatible surfaces on synthetic materials.

[Study on CH50 levels in factor D-depleted serum].

It is generally accepted that levels of serum whole complement activity (CH50) reflect the activities of complement (C) components of the classical C pathway (CP), since CH50 is assayed by use of sensitized sheep erythrocytes (EA). However, the alternative C pathway (AP) is considered to be also activated simultaneously in the process of activation of serum CP by EA. Thus, serum CH50 levels may possibly reflect not only CP but also AP activation in CH50 assay. We studied on the influence of AP activation during CH50 assay on CH50 levels, by comparison of CH50 levels in serum samples before and after treatment of factor D depletion. Polystyrene beads carrying polyanion, poly (2-acrylamide 2-methylpropane sulfonate) (PAMPS-beads), on the surface were prepared and used for preparation for factor D-depleted serum. After treatment of pooled normal human serum (NHS) with PAMPS-beads (2.5 mg/ml of serum), serum ACH50 level decreased to be undetectable, indicating that AP activation is prohibited in PAMPS-beads-treated serum. When isolated factor D was added to this PAMPS-beads-treated serum, ACH50 level recovered to that of before treatment. Immunoblot analysis revealed that factor D band observed in NHS disappeared completely after PAMPS-beads treatment. From these results, it is clear that factor-D deficient serum is prepared by PAMPS-beads treatment. Besides, since serum CH50 level was not decreased by PAMPS-beads treatment, it may be concluded that CH50 level is not affected by AP activation during CH50 assay.

The identification of a novel splicing mutation in C1qB in a Japanese family with C1q deficiency: a case report.

C1q deficiency is a rare disease that is associated with a high probability of developing systemic lupus erythematosus. We report a 4-year-old Japanese girl who presented with fever, facial erythema, joint pain, and oral ulceration. Complement deficiencies were suspected because of her persistent hypocomplementemia and normal levels of the complement proteins C3 and C4. We identified a novel homozygous splicing mutation in the C1qB gene, c.187 + 1G > T, which is the first mutation to be confirmed in a Japanese individual. Because treatment with steroids and immunosuppressive drugs was not effective, we commenced use of fresh frozen plasma to provide C1q supplements. Currently, the patient remains almost asymptomatic, and we are attempting to control the drug dosage and administration intervals of fresh frozen plasma.

A case of infantile systemic lupus erythematosus with severe lupus nephritis and EBV infection.

Infantile systemic lupus erythematosus (iSLE) is extremely rare. Patients with iSLE usually become severely unwell and have poor prognosis. Epstein-Barr virus (EBV) infection has been implicated in the development of SLE in both adults and children. Recently, we experienced a case of iSLE with severe lupus nephritis (LN) and EBV infection. A 14-month-old Japanese boy was diagnosed with iSLE according to the American Rheumatism Association criteria. Renal biopsy showed LN classified as International Society of Nephrology/Renal Pathology Society class IV-G (A), and liver biopsy showed lupus hepatitis. Steroid pulse treatment resulted in improvement of the levels of serological markers of SLE such as double-stranded DNA and complement, but his proteinuria worsened and he developed acute nephritic-nephrotic syndrome. Monthly intravenous cyclophosphamide (IVCY) therapy dramatically reduced his proteinuria and led to complete remission (urinary protein/creatinine ratio <0.1 mg/mg), with gradual improvement in levels of serological markers. EBV antibody titers and EBV polymerase chain reaction (PCR) of peripheral blood lymphocytes suggested that the onset of iSLE might have been associated with EBV infection. At his 2-year follow-up visit, he was healthy and remained in complete remission. We conclude that IVCY treatment might be well tolerated and effective in cases of iSLE. EBV infection might play an important role in the pathogenesis of iSLE.