Overexpression of schizophrenia susceptibility factor human complement C4A promotes excessive synaptic loss and behavioral changes in mice.

The complement component 4 (C4) gene is linked to schizophrenia and synaptic refinement. In humans, greater expression of C4A in the brain is associated with an increased risk of schizophrenia. To investigate this genetic finding and address how C4A shapes brain circuits in vivo, here, we generated a mouse model with primate-lineage-specific isoforms of C4, human C4A and/or C4B. Human C4A bound synapses more efficiently than C4B. C4A (but not C4B) rescued the visual system synaptic refinement deficits of C4 knockout mice. Intriguingly, mice without C4 had normal numbers of cortical synapses, which suggests that complement is not required for normal developmental synaptic pruning. However, overexpressing C4A in mice reduced cortical synapse density, increased microglial engulfment of synapses and altered mouse behavior. These results suggest that increased C4A-mediated synaptic elimination results in abnormal brain circuits and behavior. Understanding pathological overpruning mechanisms has important therapeutic implications in disease conditions such as schizophrenia.

Contribution of acute-phase reaction proteins to the diagnosis and treatment of 2019 novel coronavirus disease (COVID-19).

The emergence of 2019 novel coronavirus disease (COVID-19) is currently a global concern. In this study, our goal was to explore the changing expression levels of acute-phase reaction proteins (APRPs) in the serum of COVID-19 patients and to elucidate the immunological characteristics of COVID-19. In the study design, we recruited 72 COVID-19 patients, including 22 cases of mild degree, 38 cases of moderate degree and 12 cases of severe degree. We also recruited 20 patients with community-acquired pneumonia (CAP) and 20 normal control subjects as a comparison. Fasting venous blood was taken to detect the content of complement 3 (C3), complement 4 (C4), C-reactive protein (CRP), serum amyloid A (SAA) and prealbumin (PA). When compared the COVID-19 group with the CAP and normal control groups, respectively, the mean value of CRP and SAA in the COVID-19 group (including mild, moderate and severe patients) had increased significantly (P < 0.01), whereas the mean values of C3, C4 and PA decreased (P < 0.01). For the asymptomatic or mild symptomatic patients with COVID-19, the actual aggravation of disease may be more advanced than the clinical appearances. Meanwhile, the statistical analyses indicated that the development of COVID-19 brought about a significant increase in the content of CRP and SAA (P < 0.01), and a decline in the content of C3, C4 and PA (P < 0.01). These findings suggested that the changes in the level of APRPs could be used as indicators to identify the degree and progression of COVID-19, and the significant changes might demonstrate the aggravation of disease. This study provided a new approach to improve the clinical management plan and prognosis of COVID-19.

Proarrhythmic effect of sustained EPAC activation on TRPC3/4 in rat ventricular cardiomyocytes.

The Exchange Protein directly Activated by cAMP (EPAC) participates to the pathological signaling of cardiac hypertrophy and heart failure, in which the role of Ca(2+) entry through the Transient Receptor Potential Canonical (TRPC) channels begin to be appreciated. Here we studied whether EPAC activation could influence the activity and/or expression of TRPC channels in cardiac myocytes. In adult rat ventricular myocytes treated for 4 to 6h with the selective EPAC activator, 8-pCPT (10µM), we observed by Fluo-3 confocal fluorescence a Store-Operated Ca(2+) Entry (SOCE) like-activity, which was blunted by co-incubation with EPAC inhibitors (ESI-05 and CE3F4 at 10 µM). This SOCE-like activity, which was very small in control incubated cells, was sensitive to 30-µM SKF-96365. Molecular screening showed a specific upregulation of TRPC3 and C4 protein isoforms after 8-pCPT treatment. Moreover, sustained EPAC activation favored proarrhythmic Ca(2+) waves, which were reduced either by co-incubation with EPAC inhibitors or bath perfusion with TRPC inhibitors. Our study provides the first evidence that sustained selective EPAC activation leads to an increase in TRPC3 and C4 protein expression and induces a proarrhythmic SOCE-like activity in adult rat ventricular cardiomyocytes, which might be of importance during the development of cardiac diseases.

Significance of C4d Immunostaining in Placental Chronic Intervillositis.

Deposition of the complement split product C4d is a phenomenon studied extensively as a marker for complement activation in antibody-mediated transplant rejection. C4d also is observed in placental disease processes including spontaneous abortion, infarct, and villitis of unknown origins. Massive chronic intervillositis is a rare placental abnormality associated with increased risk of growth restriction, fetal death, and recurrent fetal loss. In this study, we evaluated C4d immunostaining in placentas with accumulation of intervillous monocytes with and without villitis. Archived placentas from Kosair Children's Hospital (Louisville, KY) and Seattle Children's Hospital (Seattle, WA) were selected and divided into 4 groups, 16 cases of intervillositis with complicated pregnancy, 15 cases of uncomplicated intervillositis, 20 cases of complicated villitis, and 13 cases of uncomplicated villitis, all with varying degrees of monocytic cells in the intervillous space. Representative specimen blocks were immunohistochemically stained for C4d. The percentage of positive staining of the microvillous surface of the syncytiotrophoblast was scored by five pathologists, and the following consensus score was determined: 0  =  0% to 5%; 1  =  5% to 25%; 2  =  25% to 75%; and 3 ≥ 75%. C4d immunostain localized to the microvillous border of syncytiotrophoblast in many of the placentas. C4d staining was more strongly associated with intervillositis than with villitis (odds ratio: 6.3; confidence interval: 2.1-18.7; P  =  0.001).

Determination of reference intervals for serum complement C3 and C4 levels in Chinese Han ethnic males.

BACKGROUND: Clinical laboratory reference intervals (RIs) for serum complement C3 and C4 levels have been established in many countries but there is a lack of published data regarding normal RIs in Chinese population. We attempted to establish RIs for serum complement C3 and C4 levels in Chinese Han ethnic males. METHODS: A total of 1,234 healthy male subjects, aged 20 - 69 years, were collected from the Fangchenggang Area Male Health and Examination Survey (FAMHES). Serum complement C3 and C4 levels were measured by immunoturbidimetry. The two-sided 95-percentile RIs were calculated using parametric statistical methods. RESULTS: Serum C3 values showed normal distribution and C4 were log-normal distributed. The two-sided 95% RIs (mean +/- 2 SD) for serum C3 and C4 were 0.656 - 1.52 g/L and 0.181 - 0.561 g/L, respectively. Body Mass Index (BMI) had a significant positive association with C3 (r = 0.342) and C4 (r = 0.258), and age had a significant positive association with C4 (r = 0.117). No significant difference was found either between smoking groups or drinking groups. A significant increase with BMI was found both for C3 (p < 0.001) and C4 (p < 0.001). BMI-specific RIs were also calculated. CONCLUSIONS: The RIs for serum C3 and C4 show a slight deviation compared to previously reported reference levels. BMI-specific reference values should be implemented in clinical laboratories.

Pharmacological and pharmacokinetic studies with vitamin D-loaded nanoemulsions in asthma model.

Vitamin D (VD) was studied for its anti-inflammatory activities with prepared VD-loaded nanoemulsions (VDNM) in ovalbumin-induced asthmatic mice in this paper. In this study, we prepared VDNM for the delivery of VD from the established composition of solid self-emulsifying drug delivery systems (sSEDDS) by spray-drying technique and evaluated its bioavailability (BA) and anti-inflammatory activities in experimental allergic asthma. After the mice were treated orally with VD or VDNM, the plasma 25(OH) D levels, polymorphonuclear cells, tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL-1ß), total antioxidant activity, and C3 and C4 complement protein levels were studied, respectively. Treatment with VDNM reduced MPO activity, oxidative stress, C3 protein level, O2(-) level as well as the production of IL-1ß and TNF-α. Pharmacokinetic studies showed that a significant increase in the maximum concentration (Cmax) and AUC0→24 h were observed in VDNM group when compared with VD group (P < 0.01). The result revealed that VDNM led to an improvement in oral BA of VD in a murine ovalbumin-induced asthma model. These data provided an important proof that VDNM might be a new potential therapy for the management of asthma in humans.

Hepatitis C virus impairs natural killer cell-mediated augmentation of complement synthesis.

UNLABELLED: Natural killer (NK) cells and the complement system play critical roles in the first line of defense against pathogens. The synthesis of complement components C4 and C3 is transcriptionally downregulated by hepatitis C virus (HCV) core and NS5A proteins, and this negative regulation is apparent in chronically HCV-infected patients. In this study, we have examined the potential contribution of an NK cell line as a model in regulating complement synthesis. Coculture of NK cells (NK3.3) with human hepatoma cells (Huh7.5) expressing HCV core or NS5A protein led to a significant increase in C4 and C3 complement synthesis via enhanced specific transcription factors. Reestablishment of complement protein expression was found to be mediated by direct interaction between NKG2D on NK cells and the hepatocyte protein major histocompatibility complex class I-related chains A and B (MICA/B) and not to be associated with specific cytokine signaling events. On the other hand, C4 and C3 synthesis remained impaired in a coculture of NK cells and Huh7.5 cells infected with cell culture-grown HCV. The association between these two cell types through NKG2D and MICA/B was examined further, with MICA/B expression in HCV-infected hepatocytes found to remain inhibited during coculture. Further experiments revealed that the HCV NS2 and NS5B proteins are responsible for the HCV-associated decrease in MICA/B. These results suggest that HCV disables a key receptor ligand in infected hepatoma cells, thereby inhibiting the ability of infected cells to respond to stimuli from NK cells to positively regulate complement synthesis. IMPORTANCE: The complement system contributes to the protection of the host from virus infection. However, the involvement of complement in viral hepatitis has not been well documented. Whether NK cells affect complement component expression in HCV-infected hepatocytes remains unknown. Here, we have shown how HCV subverts the ability of NK cells to positively mediate complement protein expression.

Positive C4d immunostaining of placental villous syncytiotrophoblasts supports host-versus-graft rejection in villitis of unknown etiology.

ABSTRACT Chronic villitis of unknown etiology (VUE) occurs in 5% of placentas submitted to pathology and is characterized by lymphohistiocytic infiltration of chorionic villi. VUE is associated with fetal growth restriction, preterm birth, and recurrent pregnancy loss. Accumulating evidence indicates that VUE may represent a host-versus-graft reaction analogous to transplant rejection. Pathologists routinely screen for antibody-mediated rejection in transplant biopsies by immunostaining for C4d, which highlights the recognition of donor cells by the host immune system. Since the hemochorial placenta is bathed in maternal blood, we hypothesized that cases of VUE may show C4d deposition onto villous syncytiotrophoblasts (STB). Chronic villitis was diagnosed in 82 of 1986 (4%) singleton placentas submitted to our department from 2007 through 2011. Forty randomly selected cases were gestational age-matched with 40 negative controls. Patient charts were reviewed and representative placental sections were immunostained for C4d. A positive C4d result was defined as circumferential immunostaining of the STB around at least one villous, or strong staining of fetal endothelial cells in the chorionic plate or stem villi. Our data indicate that VUE usually occurs in the 3rd trimester (37 ± 0.5 weeks) and is associated with significantly reduced placental weight (P  =  0.006). Positive C4d staining of STB was more common in VUE (35/40, 88%) compared with negative controls (2/40, 5%) (P < 0.0001). It was also more common in multiparous (35/66, 53%) than primiparous (2/14, 14%) women (P < 0.01). Although the precise mechanism remains to be determined, our data support the hypothesis that VUE may represent host-versus-graft rejection by the mother.

Human leukocyte antigen-specific antibodies and gamma-interferon stimulate human microvascular and glomerular endothelial cells to produce complement factor C4.

BACKGROUND: The role of the complement system in antibody-mediated rejection has been investigated in relation to circulating complement interacting with renal microvascular endothelium, resulting in the formation of peritubular capillary C4d. However, the possible importance of local complement synthesis is less clear. The aim of this study was to determine whether human vascular endothelium could produce C4 in response to stimulation in vitro. METHODS: Human microvascular endothelial cells and glomerular endothelial cells were stimulated with endotoxins, cytokines, and human leukocyte antigen-specific antibodies. Synthesis of complement was investigated using western blotting and indirect immunofluorescence. De novo C4 synthesis was confirmed by using C4 small interfering RNA. RESULTS: Glomerular and microvascular endothelium, both produce C3 and C4 complement protein. Complement synthesis was stimulant-specific-C3 was produced mainly after stimulation with lipopolysaccharide whereas C4 synthesis occurred on treatment with gamma interferon. Culture with human leukocyte antigen-specific antibodies resulted in a significant increase of C4 protein synthesis by both cell lines. CONCLUSIONS: We have shown for the first time that human microvascular endothelium can be stimulated to synthesize C4 in vitro. The implications of this for clinical transplantation, especially in the context of antibody-mediated rejection, its histological interpretation and as a potential target for therapy would have to be determined by further studies.

Pathogenesis of Sjögren's syndrome: what we know and what we should learn.

Sjögren's syndrome (SS) or autoimmune epithelitis is a prototype autoimmune disorder with unique features: a broad clinical spectrum that extends from local exocrinopathy to systemic disease and lymphoma development, and an easy access to the inflamed tissues (minor salivary glands; MSG), which enables the investigators to study the autoimmune processes. The autoimmune lesion consists of lymphocytic infiltrates that develop around the ducts and vary in severity and composition. T cells (mainly CD4(+)) are the dominant lymphocytes in mild MSG lesions, whereas B cells in severe ones. Th1 cytokines predominate in SS infiltrates, albeit Th2 and Th17 responses have been also reported. Notably, increased infiltration by IL-18(+) cells has been associated with parotid gland enlargement and C4-hypocomplementemia, which are adverse prognostic factors for lymphoma development. Even though SS pathogenesis has not been fully revealed, several aspects have been delineated. Among them, the key role of MSG epithelia in the initiation and perpetuation of local autoimmune responses is well-established and involves the capacity of epithelial cells to mediate the recruitment, homing, activation, proliferation and differentiation of immunocytes. In addition, genetic features, including certain HLA phenotypes and polymorphisms in genes encoding cytokines or factors implicated in cytokine signaling, environmental (such as viruses) and hormonal factors are thought to participate in disease pathogenesis. Herein, the known aspects of SS pathogenesis, as well as unmet issues are discussed.

Effects of acute and chronic sleep loss on immune modulation of rats.

Sleep deprivation is now recognized as an increasingly common condition inherent to modern society, and one that in many ways, is detrimental to certain physiological systems, namely, immune function. Although sleep is now viewed by a significant body of researchers as being essential for the proper working of a host of defense systems, the consequences of a lack of sleep on immune function remains to be fully comprehended. The aim of the current study was to investigate how paradoxical sleep deprivation (PSD) for 24 and 96 h and sleep restriction (SR) for 21 days by the modified multiple-platform method, and their respective 24-h recovery periods, affect immune activation in rats. To this end, we assessed circulating white blood cell counts, lymphocyte count within immune organs, as well as Ig and complement production. The data revealed that PSD for 96 h increased complement C3 and corticosterone concentration in relation to the control group. In contrast, the spleen weight, total leukocytes, and lymphocytes decreased during SR for 21 days when compared with the control group, although production of a certain class of immunoglobulin, the IgM, did increase. After recovery sleep, lymphocyte count in axillary lymph nodes grew when rats had rebound sleep after PSD for 24 h, neutrophils increased after PSD 96 h and lymphocytes numbers were higher after SR 21 days. Such alterations during sleep deprivation suggest only minor alterations of nonspecific immune parameters during acute PSD, and a significant impairment in cellular response during chronic SR.

The rat expresses two complement factor C4 proteins, but only one isotype is expressed in the liver.

The complement component C4 is well known for its complex genetics in human and mouse where it is part of a tandemly duplicated module. For the rat, no such information had been available until recently. A C4 gene duplication could be identified also in the rat, but the duplicated module maps approximately 200 kb centromerically from the canonical C4-1 gene. In this study, we present the genomic organization of the two C4 gene-containing modules and the expression of the two C4 genes in the rat (Rattus norvegicus). The duplicated module contains an intact C4 gene as well as Cyp21 and Stk19 pseudogenes. Quantitative mRNA expression analyses revealed that both C4 genes are transcribed in various organs and tissues, but displaying ample differences of C4-1 and C4-2 expression. Most notably, C4-2 is not expressed in the liver. At variance to the mouse, the expression of the rat C4 genes does not exhibit any sex dependency. By using two-dimensional gel electrophoresis and mass spectrometry, products of both C4 genes could be identified in rat serum samples. These two rat C4 isotypes are nearly identical, but differ in a functionally important amino acid residue that is known to influence the functional properties of the C4 isotypes in human.

Regulation of complement C3 and C4 synthesis in human peritoneal mesothelial cells by peritoneal dialysis fluid.

Although complement is activated in the peritoneal cavity during chronic peritoneal dialysis (PD), little is known about its role in peritoneal defence and injury related to long-term PD. We examined the impact of glucose and commercial peritoneal dialysis solutions on complement expression in HPMCs obtained by primary culture from omental tissues of consented patients undergoing elective abdominal surgery. Constitutive expression of C3 and C4 mRNA in HPMCs was up-regulated upon exposure to 75 mm glucose in a time-dependent manner. C3 and C4 protein was secreted in both apical and basolateral directions. Glucose doses beyond 100 mm markedly down-regulated C3 and C4 expression, and stimulated LDH release dose-dependently. Such cytotoxic effects were attenuated using equivalent doses of mannitol instead of glucose. Treatment with conventional lactate-buffered dialysis solution gave rise to down-regulation of C3 and C4 expression, and heightened LDH release in HPMCs. These effects correlated with the glucose strength of the solution, persisted despite replacement with a bicarbonate-buffered solution, aggravated by glycated albumin, and were partially abrogated by supplementation with 10% fetal bovine serum in the culture system. Our findings suggest that the artificial conditions imposed by PD lead to alterations in local complement synthesis that have implications for the role of the peritoneal mesothelium in both inflammation and defence.

Significance of postoperative adjuvant immunochemotherapy after curative resection of colorectal cancers: identification of responders incorporating the age factor.

To identify responders when protein-bound polysaccharide (PSK) is used in adjuvant immunochemotherapy after curative resection of colorectal cancers, we examined the host and tumor factors that affect the prognosis incorporating the age factor. A total of 101 patients who had undergone macroscopic curative resection of colorectal cancer were treated with mitomycin C + fluoropyrimidine oral antineoplastics + PSK (MFP therapy) for two years in principle. These cases were divided into two age groups of <65 years [n=55; 54.8 +/- 8.3 years (mean +/- SD)] and > or =65 years (n=46; 69.1 +/- 3.3 years). Host factors including humoral factors (complement C3 and C4), immunosuppressive acidic protein (IAP), lymphocyte transformation (cellular factors) induced by various mitogens [phytohemagglutinin (PHA), pokeweed mitogen (PWM), and PSK], and tumor markers (CEA, CA19-9) were measured. The cases were divided by the cut-off value of each parameter into > or = cut-off value and < cut-off value groups, and the 5-year survival rates were compared. The cut-off values obtained for these parameters and the tumor factor (Dukes class) were subjected to multivariate analysis to identify the markers that affect prognosis. The 5-year mortality rate was 74.5% in the <65 age group and 56.8% in the > or =65 age group, with a tendency of better prognosis in the <65 age group (p=0.1109). Compared to the <65 age group, the > or =65 age group showed higher levels of C3 (2-way ANOVA: p=0.0582), C4 (p=0.0009) and IAP (p=0.0110) over time, but lower PSK-induced stimulation index (SI) as an indicator of cellular immunity) (p=0.0001) and PHA-induced SI (p=0.2650) over time. These results indicated that compared to patients aged <65 years, patients aged > or =65 years were characterized by lowered cellular immunity in addition to augmented complement production and an aggravated immunosuppressive state, suggesting the presence of some differences in host immune function with aging. Using the Cox proportional hazard model, the prognostic determinant was found to be Dukes C in the <65 age group, and CEA level in the > or =65 age group. The present results suggested that analysis of prognostic determinants of this therapy should take into account the age factor. Especially in elderly subjects, responders to PSK may be identified using the preoperative CEA value.

Formation of high affinity C5 convertase of the classical pathway of complement.

C3/C5 convertase is a serine protease that cleaves C3 and C5. In the present study we examined the C5 cleaving properties of classical pathway C3/C5 convertase either bound to the surface of sheep erythrocytes or in its free soluble form. Kinetic parameters revealed that the soluble form of the enzyme (C4b,C2a) cleaved C5 at a catalytic rate similar to that of the surface-bound form (EAC1,C4b,C2a). However, both forms of the enzyme exhibited a poor affinity for the substrate, C5, as indicated by a high Km (6-9 microM). Increasing the density of C4b on the cell surface from 8,000 to 172,000 C4b/cell did not influence the Km. Very high affinity C5 convertases were generated only when the low affinity C3/C5 convertases (EAC1,C4b,C2a) were allowed to deposit C3b by cleaving native C3. These C3b-containing C3/C5 convertases exhibited Km (0.0051 microM) well below the normal concentration of C5 in blood (0.37 microM). The data suggest that C3/C5 convertase assembled with either monomeric C4b or C4b-C4b complexes are inefficient in capturing C5 but cleave C3 opsonizing the cell surface with C3b for phagocytosis. Deposition of C3b converts the enzymes to high affinity C5 convertases, which cleave C5 in blood at catalytic rates approaching Vmax, thereby switching from C3 to C5 cleavage. Comparison of the kinetic parameters with those of the alternative pathway convertase indicates that the 6-9-fold greater catalytic rate of the classical pathway C5 convertase may compensate for the fewer numbers of C5 convertase sites generated upon activation of this pathway.

The effect of locally synthesised complement on acute renal allograft rejection.

The complement system of components and receptors is one of the earliest forms of defence. Excessive or inappropriate activation can result in tissue damage, classically illustrated in immune-mediated nephritis. In addition, complement forms a bridge between innate and adaptive immunity, helping to prepare and focus T and B lymphocyte responses. More recent research in renal allograft models has shown that complement-inhibited and complement-deficient animals have reduced inflammatory injury and lowered antidonor immune responses. Furthermore, it is known that the transplanted kidney is a significant site of local synthesis of C3, although until recently the relative contribution of locally produced C3 to transplant injury was unknown. Current evidence indicates that defective local synthesis of C3 both reduces tissue injury and lowers the antidonor T cell response, substantially increasing graft survival. Among various possible explanations to account for these findings, the data favours a direct effect of complement on alloreactive T cell stimulation. Study of complement gene regulation by common immunosuppressive agents suggests that they do not influence local complement synthesis. Alternative approaches are therefore required to control the local effect of complement in the extravascular tissue compartment of the graft.

Studies on the mechanisms of allergen-induced activation of the classical and lectin pathways of complement.

Allergen extracts are efficient activators of the complement system trough the classical pathway. Involvement of the lectin pathway was not previously studied. To further examine the mechanism of complement activation by allergens, in vitro experiments, which covered early steps both of classical and lectin pathways, were performed. Two types of allergens used in these studies: parietaria (PA) and house dust (HD) mite extracts. These allergen extracts bound to the globular head of C1q and interacted with purified mannan-binding lectin (MBL) as measured by solid-phase ELISA. None of the allergen extracts was able to activate human C1 in vitro, as measured by the determination of the split products of C1s in a reconstituted precursor C1 preparation. Neither the HD nor the PA extracts induced C4d generation above background in the serum of three subjects with hypogammaglobulinaemia but normal complement haemolytic activity. After reconstitution to normal level with purified human IgG, allergen extracts induced C4d formation above control at a level comparable to that measured in normal serum incubated with the same amounts of the extracts. HD-induced C4d generation was about the same comparable in MBL-depleted serum and in normal sera. In contrast PA induced no C4d formation in the MBL-depleted serum, whereas reconstitution with purified MBL restored C4d generation. These in vitro findings indicate that although the allergen extracts can bind purified C1q and MBL, they require IgG for efficient complement activation. Depending on the allergens, this activation may be initiated through C1, MBL, or both.

Macrophage-derived complement component C4 can restore humoral immunity in C4-deficient mice.

Mice with a disrupted C4 locus (C4(-/-)) have an impaired immune response to thymus-dependent Ags. To test the role of bone marrow-derived C4 in humoral immunity, we reconstituted deficient animals with wild-type bone marrow or an enriched fraction of bone marrow-derived macrophages. C4 chimeras were immunized with 4-hydroxy-3-nitrophenyl(5) conjugated to keyhole limpet hemocyanin (NP(5)- KLH) or infected with HSV-1, and the Ab response was evaluated. Wild-type bone marrow rescued the humoral immune response to both Ags, i.e., the soluble Ag and HSV-1, demonstrating that local C4 production is sufficient for humoral responses. Although the C4 chimeric animals lacked detectable C4 in their sera, C4 mRNA was identified in splenic sections by in situ hybridization, and C4 protein deposits were identified in the germinal center areas of splenic follicles by immunofluorescence staining. Macrophages derived from bone marrow produced sufficient C4 protein to restore the humoral response to NP(5)-KLH in C4-deficient animals when administered along with Ag. Cell-sorting experiments, followed by C4-specific RT-PCR, identified splenic macrophages (CD11b(+), CD11c(-)) as a cellular source for C4 synthesis within the spleen.

Complement activation in chromosome 13 dementias. Similarities with Alzheimer's disease.

Chromosome 13 dementias, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with neurodegeneration and cerebrovascular amyloidosis, with striking neuropathological similarities to Alzheimer's disease (AD). Despite the structural differences among the amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD), these disorders are all characterized by the presence of neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with markers of glial activation, suggestive of local inflammation. Proteins of the complement system and their pro-inflammatory activation products are among the inflammation markers associated with AD lesions. Immunohistochemistry of FBD and FDD brain sections demonstrated the presence of complement activation components of the classical and alternative pathways as well as the neo-epitope of the membrane attack complex. Hemolytic experiments and enzyme-linked immunosorbent assays specific for the activation products iC3b, C4d, Bb, and C5b-9 indicated that ABri and ADan are able to fully activate the complement cascade at levels comparable to those generated by Abeta1-42. ABri and ADan specifically bound C1q with high affinity and formed stable complexes in physiological conditions. Activation proceeds approximately 70-75% through the classical pathway while only approximately 25-30% seems to occur through the alternative pathway. The data suggest that the chronic inflammatory response generated by the amyloid peptides in vivo might be a contributing factor for the pathogenesis of FBD and FDD and, in more general terms, to other neurodegenerative conditions.

Complement activated C4d immunoreactive oligodendrocytes delineate small cortical plaques in multiple sclerosis.

C4d-immunoreactive complement-activated oligodendrocytes (C4d-CAOs) have been described in several neurodegenerative diseases but have not been studied in multiple sclerosis (MS). Here we report that such CAOs delineate miniature MS plaques of 300-500 mum diameter. They are devoid of myelin and are surrounded by a rim of activated microglia intermingled with the C4d-CAOs. Although C4d-immunostained periaxonal oligodendroglial processes are often swollen, the axons of passage appear undamaged and extend through the demyelinated plaque area. No immunostaining with other components of the complement cascade (C1q-C9) was observed in association with these miniature plaques. However, in large MS lesions, C1q-C9 immunoreactive fibers were present, indicating complete activation of the complement cascade in these more developed lesions. It is possible that the miniature plaques, bordered by C4d-CAOs, represent the earliest stage of plaque development, preceding even the larger, transient plaques frequently observed in serial MRI studies. The association of CAOs with miniature areas of demyelination suggests a direct attack on oligodendroglial cells by the early complement components as an initiating event in MS. Incomplete complement activation indicates that this step may be reversible, whereas full and persistent activation as observed in large MS lesions may lead to death of oligodendroglia with permanent axonal damage.