Activation of the lectin complement pathway in post-streptococcal acute glomerulonephritis

The aim of the present study was to elucidate the correlation between complement pathways and clinicopathological findings in post-streptococcal acute glomerulonephritis (PSAGN). Immunohistological staining was performed on renal specimens obtained from 18 patients with PSAGN and 20 controls, using antibodies against IgG, IgA, IgM, C1q, C3c, C4, fibrinogen, factor B, C4-binding protein (C4-bp), C5b-9, CD59, mannose-binding lectin (MBL) and MBL-associated serine protease-1 (MASP-1). Controls showed no deposition of any antibody. In seven patients, glomerular deposits of C3c, C4, factor B, C4-bp, C5b-9, CD59, MBL and MASP-1 were found. In the remaining 11 patients, glomerular deposits of neither C4 nor MBL/MASP-1 were found, and glomerular deposits of C3c, factor B, C5b-9 and CD59 were evident. C4-bp was detected in seven of these 11 patients. Glomerular deposits of fibrinogen were detected in five of seven patients with MBL/MASP-1 deposits and in only two of 11 patients without MBL/MASP-1 deposits. Hematuria was prolonged in three of seven patients with MBL/MASP-1 deposits through follow up, whereas urinalysis was normal in all patients without MBL/MASP-1 deposits. However, the histological indicators were not different between the two groups. To the authors' knowledge this is the first report to show that complement activation through both the alternative and lectin pathways is evident in some patients with PSAGN. Complement activation is promoted in situ in the glomerulus.     

补体活化的凝集素途径在IgA肾病发病中的作用

目的探讨补体活化的凝集素途径在IgA肾病发病中的作用。方法用ELISA方法检测20例IgAN患儿和正常儿童血清的MBL水平,免疫组化检测53例IgA肾病、23例非IgA沉积肾小球疾病患儿肾组织甘露聚糖结合凝集素(MBL)、MBL相关的丝氨酸蛋白酶(MASP-1)、C3、C1q、IgA和C5b-9的表达,了解有无MBL-MASP-1途径补体活化参与IgAN发病;回顾性分析MBL-MASP-1沉积阳性组和MBL-MASP-1沉积阴性组患儿不同临床表现的差异;分析IgAN的MBL沉积强度与病理分级的相关性,了解不同程度补体活化与IgAN病理损害的关系。结果 22例IgAN患儿肾小球有MBL、MASP-1、C3和C5b-9沉积,23例非IgA沉积肾脏疾病患儿均无MBL和MASP-1沉积;IgA肾病患儿血清MBL水平和对照组统计学无明显的差异;系膜区MBL沉积强度与IgA沉积强度之间存在正相关关系(rs=0.865),但与IgAN病理分级无关;MBL-MASP沉积阳性组患儿蛋白尿水平显著高于MBL-MASP沉积阴性组,肉眼血尿更为多见。结论局部凝集素途径的补体活化在IgAN发病中有重要作用;MBL-MASP沉积阳性的IgAN患儿蛋白尿水平更高,肉眼血尿更为多见。     

Mannose-binding lectin in innate immunity: past, present and future

The human collectin, mannose-binding lectin (MBL), is an important protein of the humoral innate immune system. With multiple carbohydrate-recognition domains, it is able to bind to sugar groups displayed on the surfaces of a wide range of microorganisms and thereby provide first-line defence. Importantly, it also activates the complement system through a distinctive third pathway, independent of both antibody and the C1 complex. Three single point mutations in exon 1 of the expressed human MBL-2 gene appear to impair the generation of functional oligomers. Such deficiencies of functional protein are common in certain populations, e.g. in sub-Saharan Africa, but virtually absent in others, e.g. indigenous Australians. MBL disease association studies have been a fruitful area of research and implicate a role for MBL in infective, inflammatory and autoimmune disease processes. Overall, there appears to be a genetic balance in which individuals generally benefit from high levels of the protein. However, in certain situations, reduced levels of circulating MBL may be beneficial to the host and this may explain the persistence of the deleterious gene polymorphisms in many population groups.     

Exon structure of the gene encoding the human mannose-binding protein-associated serine protease light chain: comparison with complement C1r and C1s genes

Mannan or mannose-binding protein (MBP) requires a novel serineprotease termed MBP-associated serine protease (MASP) for activationof the complement cascade. In this study, we analyzed MASP genomicclones and found that the light chain (catalytic domain) isencoded by six exons, whereas those of the complement C1 subunits,C1r and C1s, and the haptoglobin segment have been reportedto be encoded by a single exon. We confirmed the intron-lackingsequence of C1r by analysis of its genome. These results, inconjunction with those obtained by constructing a phylogenetictree for these proteins, suggest that the MASP gene is a prototypeand that the Intron-lacking sequences of the other serine proteaseshave a more recent history.     

The lectin pathway of complement activation contributes to protection from West Nile virus infection

The function of the lectin pathway of complement activation in vivo against West Nile virus (WNV) or many other pathogenic viruses has not been defined. Mice deficient in lectin pathway recognition molecules (mannose binding lectin-A (MBL-A) and mannose binding lectin-C (MBL-C)) or the effector enzyme mannan-binding lectin-associated serine protease-2 (MASP-2), were more vulnerable to WNV infection than wild type mice. Compared with studies of mice deficient in factors of the classical or alternative pathway, MBL-A^−/− × MBL-C^−/− or MASP-2^−/− mice showed a less severe course of WNV infection. Indeed, a deficiency in lectin pathway activation did not significantly affect the kinetics of viral spread to the central nervous system (CNS) nor did it profoundly alter generation of adaptive B and T cell immune responses. We conclude that MBL-mediated recognition and lectin pathway activation have important yet subordinate functions in protecting against WNV infection and disease.     

Clinicopathological features of cryoglobulinemic glomerulonephritis associated with HBV infection: a retrospective analysis of 8 cases in China

Aims: We retrospectively analyzed clinicopathologic features of 8 cases of hepatitis B virus-associated glomerulonephritis with hyaline thrombi, to confirm the diagnosis of cryoglobulinemic glomerulonephritis (CRYGN) associated with HBV infection. Methods: Retrospective analysis was carried out with demographic information, clinical manifestations, laboratory parameters, pathological and prognostic features. Results: The median age of 8 patients was 30.5 years (range, 21-75 years), including 6 males and 2 femles (M:F = 3:1). One patient had Raynaud’s syndrome. Cryoglobulin testing was performed in 4 cases of our series, and 3 cases had elevated cryocrit (>256). Serum C4 decreased in all detected cases. Histopathologically, all cases showed hyaline thrombi occluded in capillary lumina; Co-deposit of IgG, IgM, IgA, Fib, C3d, C4d, C1q, HBsAg and HBcAb were identified in hyaline deposit/hyaline thrombi with polyclonal Igκ and Igλ staining. Ultrastructural examination confirmed the hyaline thrombi to be huge electron-dense bodies, which were a homogeneous texture. Conclusions: The results suggest that 8 cases in the series are CRYGN associated with HBV infection. The incidence of CRYGN associated HBV was extremely low. Our series suggested that prognosis of CRYGN associated HBV was better in patients with mild symptoms, but it was poor in elder patients with severe vasculitis.     

Systemic sclerosis and cryoglobulinemia: Our experience with overlapping syndrome of scleroderma and severe cryoglobulinemic vasculitis and review of the literature

Objective

Systemic sclerosis (SSc) is an immune-mediated disorder characterized by multiple organ fibrotic alterations and diffuse microangiopathy. The SSc can be associated with other connective tissue diseases and less frequently with systemic vasculitides, including cryoglobulinemic vasculitis (CV). The aim of the present study was to investigate the prevalence of CV in a large series of SSc patients.

Methods

The presence of serum cryoglobulins was detected in 246 SSc patients (24 M and 222 F, age 61 ± 13.5 SD years, disease duration 9.3 ± 6.7 SD years); the observed clinico-serological findings, in particular the presence of SSc–CV overlapping syndrome, were carefully analyzed and compared with previous data reported in the literature.

Results

The presence of circulating cryoglobulins was found in 7/246 (2.8%) of SSc patients; namely, 2 subjects only trace amounts of cryoglobulins, while 5 (2%) showed mixed cryoglobulinemia (type II, IgG-IgMk), low C4, rheumatoid factor seropositivity, and hepatitis C virus infection. Among SSc patients with serum mixed cryoglobulins, 4 (1.6%) developed a clinically overt CV, while the other one was totally asymptomatic with regard to typical vasculitic manifestations. Patients with SSc–CV overlapping syndrome had limited cutaneous SSc with serum anticentromere antibodies, pulmonary hypertension, clinico-serological features of HCV-related CV, and non-healing skin ulcers of the lower limbs. In all cases, the diagnosis of SSc preceded the clinical onset of CV, from 3 to 17 years. The treatment with rituximab was useful on skin ulcers of lower limb in 2/3 patients; however, the overall clinical outcome of the four SSc–CV patients was unusually severe: one with very severe skin ulcers complicated by gangrene required bilateral through-the knee amputation, the other three subjects died because of severe heart failure, and in two cases because of untreatable pulmonary hypertension.In the literature, the prevalence of mixed cryoglobulinemia in scleroderma patients is quite rare (range 0.3–2%); while, the association of SSc with clinically overt CV is only anecdotally described, always in the absence of HCV infection.

Conclusion

The SSc–CV overlapping syndrome described here is characterized by markedly severe vascular manifestations responsible for very poor prognosis; these peculiar clinical manifestations suggest a synergic activity of typical scleroderma microangiopathy and cryoglobulinemic vasculitis.     

Stringent regulation of complement lectin pathway C3/C5 convertase by C4b-binding protein (C4BP)

The complement lectin pathway, an essential component of the innate immune system, is geared for rapid recognition of infections as each C4b deposited via this pathway is capable of forming a C3/C5 convertase. In the present study, role of C4b-binding protein (C4BP) in regulating the lectin pathway C3/C5 convertase assembled on zymosan and sheep erythrocytes coated with mannan (E_Man) was examined. While the C4BP concentration for inhibiting 50% (IC₅₀) formation of surface-bound C3 convertase on the two surfaces was similar to that obtained for the soluble C3 convertase (1.05 nM), 3- and 41-fold more was required to inhibit assembly of the C5 convertase on zymosan (2.81 nM) and E_Man (42.66 nM). No difference in binding interactions between C4BP and surface-bound C4b alone or in complex with C3b was observed. Increasing the C4b density on zymosan (14,000–431,000 C4b/Zym) increased the number of C4b bound per C4BP from 2.87 to 8.23 indicating that at high C4b density all seven α-chains of C4BP are engaged in C4b-binding. In contrast, the number of C4b bound per C4BP remained constant (3.79 ± 0.60) when the C4b density on E_Man was increased. The data also show that C4BP regulates assembly and decay of the lectin pathway C3/C5 convertase more stringently than the classical pathway C3/C5 convertase because of a 7- to 13-fold greater affinity for C4b deposited via the lectin pathway than the classical pathway. C4BP thus regulates efficiently the four times greater potential of the lectin pathway than the classical pathway in generating the C3/C5 convertase and hence production of pro-inflammatory products, which are required to fight infections but occasionally cause pathological inflammatory reactions.     

Mannan-binding lectin MBL2 gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.     

Effects of MASP2 haplotypes and MASP‐2 levels in hepatitis C‐infected patients

Mannan‐binding lectin (MBL) and MBL‐associated serine protease 2 (MASP‐2) are components of the lectin pathway, which activate the complement system after binding to the HCV structural proteins E1 and E2. We haplotyped 11 MASP2 polymorphisms in 103 HCV patients and 205 controls and measured MASP‐2 levels in 67 HCV patients and 77 controls to better understand the role of MASP‐2 in hepatitis C susceptibility and disease severity according to viral genotype and fibrosis levels. The haplotype block MASP2*ARDP was associated with protection against HCV infection (OR = 0.49, p = .044) and lower MASP‐2 levels in controls (p = .021), while haplotype block AGTDVRC was significantly increased in patients (OR = 7.58, p = .003). MASP‐2 levels were lower in patients than in controls (p < .001) and in patients with viral genotype 1 or 4 (poor responders to treatment) than genotype 3 (p = .022) and correlated inversely with the levels of alkaline phosphatase, especially in individuals with fibrosis 3 or 4 (R = ?.7, p = .005). MASP2 gene polymorphisms modulate basal gene expression, which may influence the quality of complement response against HCV. MASP‐2 levels decrease during chronic disease, independently of MASP2 genotypes, most probably due to consumption and attenuation mechanisms of viral origin and by the reduced liver function, the site of MASP‐2 production.     

Plasma-derived mannose-binding lectin shows a direct interaction with C1-inhibitor

MBL-deficiency has been associated with an increased frequency and severity of infection, in particular in children and under immunocompromized conditions. In an open uncontrolled safety and pharmacokinetic MBL-substitution study using plasma-derived MBL (pdMBL) in MBL-deficient pediatric oncology patients, we found that despite MBL trough levels above 1.0 μg/ml MBL functionality was not efficiently restored upon ex vivo testing.     

慢性丙型肝炎患者血浆内毒素和血清MBL变化及临床意义

目的:探讨了慢性丙型肝炎患者血浆内毒素和血清甘露聚糖结合凝集素(MBL)水平的变化及意义.方法:应用鲎试验和酶联法对31例慢性丙型肝炎患者进行了血浆内毒素和血清MBL测定,并与35名正常健康人作比较.结果:慢性丙型肝炎患者血浆内毒素和血清MBL水平均非常显著地高于正常人水平(P<0.01),且内毒素水平与MBL呈正相关...     

The effects of direct-acting antiviral agents on the frequency of myeloid-derived suppressor cells and natural killer cells in patients with chronic hepatitis C

Currently, hepatitis C antiviral therapy is entering a new era with the use of direct-acting antiviral (DAA) agents. However, the precise immunological influences of DAA therapy in patients with chronic hepatitis C (CHC) are insufficiently understood. This study aimed to investigate the effects of DAA therapy on the frequency of myeloid-derived suppressor cells (MDSCs), T lymphocytes, and natural killer (NK) cells in patients with CHC. Thirty-two treatment-naive CHC patients were treated with DAA therapy, and the frequency of immune cells was analyzed by flow cytometry at various time points during and after therapy. Sixteen healthy donors were recruited for comparison. DAA therapy decreased the frequency of MDSCs and monocytic MDSCs in patients with CHC to a normal level. DAA therapy also increased the CD8⁺ T and NK cell levels in patients with CHC. In addition, activation (NKp30 and NKp46) and inhibitory (NKG2A) receptors on NK cells were downregulated to yield an NK cell phenotype resembling that observed in the healthy controls. This study provides insight into the normalization of immune cell levels under DAA therapy and indicates that restoration of the immune system in patients with CHC strongly supports long-term curative hepatitis C virus eradication.     

Severe fibrosis in hepatitis C virus-infected patients is associated with increased activity of the mannan-binding lectin (MBL)/MBL-associated serine protease 1 (MASP-1) complex

Mannan-binding lectin (MBL) binds microorganisms via interactions with glycans on the target surface. Bound MBL subsequently activates MBL-associated serine protease proenzymes (MASPs). A role for MBL in hepatitis C virus (HCV) infection had been indicated by previous studies examining MBL levels and polymorphisms in relation to disease progression and response to treatment. We undertook this study to investigate a possible relationship between disease progression and functional MBL/MASP-1 complex activity. A functional assay for MBL/MASP-1 complex activity was employed to examine serum samples from patients with chronic HCV infection, non-HCV liver disease and healthy controls. Intrapatient consistency of MBL/MASP-1 complex activity levels was assessed in sequential samples from a subgroup of patients. Median values of MBL/MASP-1 complex activity were higher in sera from patients with liver disease compared with healthy controls. MBL/MASP-1 complex activity levels correlate with severity of fibrosis after adjusting for confounding factors (P = 0.003). MBL/MASP-1 complex activity was associated more significantly with fibrosis than was MBL concentration. The potential role of MBL/MASP-1 complex activity in disease progression is worthy of further study to investigate possible mechanistic links.     

Resolution of HBV infection occurs sooner than recovery of renal disease in adult serum HBsAg‐negative HBV–associated glomerulonephritis

Most cases of hepatitis B virus–associated glomerulonephritis (HBV‐GN) occur in children and present with serum HBsAg positivity. Few studies have investigated adult patients with HBV‐GN who are serum HBsAg‐negative. This study aimed to determine the clinical and pathological features of adult patients with HBV‐GN who are serum hepatitis B surface antigen (HBsAg)‐negative. Clinical, pathologic, and laboratory findings were collected and analyzed in a cohort of 27 adult patients with HBV‐GN who were serum HBsAg‐negative upon diagnosis. The study population included mostly men of middle age (40‐59 years). Clinically, patients presented with nephrotic syndrome. Serum immunoglobulin G levels were low, whereas serum immunoglobulin M, immunoglobulin A, complement C3 (C3), and complement C4 (C4) levels as well as liver and renal function tests were normal in most or all patients. Among the 27 patients, 21 tested positive for HBV antibodies. Membranous nephropathy was the dominant pathological form on kidney biopsy. In addition, only a few patients showed a “full house” staining pattern and renal immune deposit of complement C1q (C1q). Serum HBsAg‐negative HBV‐GN may represent a late stage of HBV infection. We recommend routine testing for HBV markers on renal biopsy in regions where HBV is prevalent, even when tests for serum HBV markers are negative.     

Factors predisposing to the occurrence of cryoglobulinemia in two cohorts of Egyptian and Japanese patients with chronic hepatitis C infection: ethnic and genotypic influence

The association between cryoglobulinemia and hepatitis C virus (HCV) infection has been reported. However, the factors underlying its wide variation of occurrence have not yet been well identified. To investigate this, cryoglobulinemia was studied in four cohorts of Egyptian and Japanese patients. Fifty Egyptian patients with chronic hepatitis C, infected with genotype 4 (the predominant HCV genotype in Egypt), were compared with 50 age- and sex-matched Japanese patients, infected with HCV genotype 1b (the predominant HCV genotype in Japan). Thirty-two Egyptian and 30 age- and sex-matched Japanese patients with chronic hepatitis B were included as controls. All patients were noncirrhotic. Antinuclear antibody (ANA), immunoglobulins (Ig), and cryoglubulins were assessed. Results showed a significantly higher prevalence of cryoglobulinemia in chronic hepatitis C Japanese genotype 1b (40%) as compared with Egyptian genotype 4 (14%), P = 0.003, while no difference was found between Japanese (17%) and Egyptian chronic hepatitis B controls (13%). Symptomatic cryoglobulinemia was more prevalent in the Japanese than in the Egyptian chronic hepatitis C group (10% vs. 4%), but the difference was not statistically significant. Univariate analysis showed no association between cryoglobulinemia and either age, sex, alanine aminotransferase level, or HCV viral load in Japanese or Egyptian patients, while the mean IgM level was significantly higher in the cryoglobulin-positive than in the cryoglobulin-negative chronic hepatitis C patients in each group (P = 0.003 and 0.017, respectively). Cryoglobulinemia was found to be significantly associated with both high IgG level (P = 0.020), and positive ANA (P < 0.001) in Japanese patients with chronic hepatitis C, genotype 1b but not in Egyptians with genotype 4. Multivariate analysis showed that the only factors predisposing to cryoglobulinemia were Japanese ethnicity with HCV genotype1b (P = 0.002, OR = 2.56), high IgM level of >245 mg/dl (P = 0.018, OR = 2.05) and female gender (P = 0.040, OR = 1/0.66). In conclusion, cryoglobulinemia is prevalent in Japanese patients with chronic hepatitis C infected with genotype 1b, but cryoglobulinemia is not common in Egyptians with HCV genotype 4. Although it was not possible to evaluate ethnicity and HCV genotype separately in this study, HCV genotype 1b appears to predispose more to cryoglobulinemia than does genotype 4. Female gender and high serum IgM level were also related.     

Mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system

Blood samples were collected over a 4-year period from 335 children (aged 1-16 years) suffering from recurrent respiratory infections and 78 controls. The patients were subdivided into four groups: I, children with no immune system defects detected (n = 101); II, children with allergies (n = 94); III, children with humoral response defects (n = 93); and IV, children with disturbances of cellular immunity (n = 66). Nineteen patients had both humoral and cellular abnormalities. All patients and controls were investigated to determine the exon 1 and promoter region variants of the mbl-2 gene. MBL serum concentrations were also determined in samples from 291 patients and 75 controls. The proportion of O (B, D or C) alleles was significantly higher in the patient group compared to controls, and this association was strongest for subgroup III. The promoter LX variant frequency was also commoner in the patients as a whole, and significantly so in subgroups II and IV. Genotypes markedly influenced MBL concentrations in all groups, and correlated with ability to activate the lectin pathway of complement activation. The strongest and most significant inverse correlations between serum MBL and respiratory disease were found in patient group III and in 17 patients with multiple humoral and/or cellular abnormalities. Among nine patients with unexpectedly low LP activity in view of their MBL concentrations, one person was found to be MASP-2 deficient. Our results indicate that mannan-binding lectin insufficiency, with or without a coexisting immune defect, is associated with the occurrence of recurrent respiratory infections in childhood, and this relationship is particularly strong and statistically significant in children with concomitant impairments of humoral immunity.