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A new serological reaction in patients with polymyalgia rheumatica and/or giant cell (temporal) arteritis: deposition of complement C4 and C3 components on rat kidney structures detected by indirect immunofluorescence.

Using indirect immunofluorescence deposition of complement C4 and C3 components to rat kidney medullary structures was demonstrated. This serological reaction (C4/C3-IFT) is regularly obtained with fresh sera of patients suffering from active polymyalgia rheumatica (PMR) and/or giant cell (temporal) arteritis (GCA). Sera of normal controls and of steroid-treated PMR and/or GCA patients in clinical remission have a negative C4/C3-IFT reaction. A positive conversion of the test in steroid-treated patients indicates enhanced disease activity. Because of its technical simplicity, C4/C3-IFT can be routinely used first, as a reliable serological marker in the primary diagnosis of PMR and/or GCA and second, as a sensitive criterion of disease activity in these patients. C4/C3-IFT reactivity is, however, not specific for GCA and/or PMR. Various systemic inflammatory diseases may have a positive reaction as well (e.g., certain viral and bacterial infections, malignant tumors, vasculitides, inflammatory joint diseases of different etiology). Recent experimental findings suggest that C-reactive protein (CRP) in patients' sera mediates an activation of the classical complement pathway resulting in a deposition of C4 and C3 complement components to certain rat kidney structures.

Adult↗

Immunoglobulins in human dental caries.

The penetration and complement fixation of sIgA, IgM and IgG were studied in advanced human dental caries with double-staining immunofluorescence technique. Immunoglobulins were found in dental plaque and in the superficial layers of the lesions which were exposed to saliva. sIgA was the most frequent, followed by IgG and IgM. IgG had the greatest capacity for penetration into the lesions, followed by IgM and sIgA. IgM was most frequently found in association with complement C3.

Complement C3↗

Increased sensitivity of early apoptotic cells to complement-mediated lysis.

Opsonization of apoptotic cells with complement proteins contributes to their clearance by phagocytes. Little is known about the lytic effects of complement on apoptotic cells. Sensitivity of cells treated with anti-Fas antibody (Jurkat cells), staurosporine or etoposide (Raji cells) to lysis by complement was examined. As shown here, early apoptotic cells are more sensitive to lysis by antibody and complement than control cells. More complement C3 and C9 bound to apoptotic than to control cells, even though antibody binding was similar. Enhanced killing and C3/C9 deposition were blocked by benzyloxy-Val-Ala-Asp-fluoromethylketone, a pan-caspase inhibitor. Complement-mediated lysis of early apoptotic cells was also prevented by inhibitors of caspases 6, 8, 9 or 10. In contrast, caspase inhibitors had no effect on the lysis of non-apoptotic Jurkat and Raji cells. Early apoptotic Jurkat cells were also more sensitive to lysis by the pore formers streptolysin O and melittin. Sensitivity of Jurkat Bcl-2 transfectants to lysis by complement was analyzed. Enhanced Bcl-2 expression was associated with reduced C3 deposition and lower sensitivity to complement-mediated lysis. These results demonstrate that at an early stage in apoptosis, following caspase activation, cells become sensitive to necrotic-type death by complement and other pore formers. Furthermore, they suggest that Bcl-2 is actively protecting Jurkat cells from complement-mediated lysis.

Amino Acid Chloromethyl Ketones↗

Photodynamic therapy-generated vaccine for cancer therapy.

A target tumor-derived whole cancer cell therapeutic vaccine was developed based on an in vitro pre-treatment by photodynamic therapy (PDT) and was investigated using a poorly immunogenic tumor model. The vaccine was produced by incubating in vitro expanded mouse squamous cell carcinoma SCCVII cells for 1 h with photosensitizer benzoporphyrin derivative (BPD), then exposing to light (690 nm, 1 J/cm2) and finally to a lethal X-ray dose. Treatment of established subcutaneous SCCVII tumors growing in syngeneic C3H/HeN mice with 2 x 10(7) PDT-vaccine cells per mouse by a peritumoral injection produced a significant therapeutic effect, including growth retardation, regression and cures. Tumor specificity of this PDT-generated vaccine was demonstrated by its ineffectiveness when prepared from a mismatched tumor cell line. Vaccine cells retrieved from the treatment site at 1 h postinjection were intermixed with dendritic cells (DC), exhibited heat shock protein 70 on their surface, and were opsonized by complement C3. Tumor-draining lymph nodes treated by the PDT-vaccine contained dramatically increased numbers of DC as well as B and T lymphocytes (with enlarged memory phenotype fraction in the latter), while high levels of surface-bound C3 were detectable on DC and to a lesser extent on B cells. The PDT-vaccine produced no therapeutic benefit against tumors growing in C3-deficient hosts. It is suggested that surface expression of heat shock proteins and complement opsonization are the two unique features of PDT-treated cells securing avid immune recognition of vaccinated tumor and the development of a strong and effective antitumor adaptive immune response.

Animals↗

Antimicrobial factors and bacterial correlation in chronic otitis media with effusion.

Analyses of effusions and sera from patients with otitis media with effusion demonstrated local production in the middle ear of lysozyme, IgA and IgG. The effusion IgM was markedly elevated in some patients, also indicating local production. Complement C3 with rare exception was significantly lower in effusions than sera, suggesting utilization of complement in the middle ear, perhaps in conjunction with antibodies. The presence of high levels of lysozyme and immunoglobulins in effusions correlates with the low isolation rate of microorganisms in culture and may influence survival of organisms in the middle ear.

Child↗

Acute hemorrhagic pancreatic necrosis in mice: lack of a pathogenetic role for complement.

Feeding a choline-deficient diet containing 0.5% DL-ethionine induces an acute hemorrhagic pancreatitis in 100% of young female mice. Evidence for deposition of the third component of complement (C3) on acinar cell plasma membranes was sought, during the inductive stages, by a sandwich immunofluorescence technique. Such a localization could not be demonstrated even though the method is capable of detecting less than 8 x 10(-5) microgram of protein/mm2 of cell membrane. Artifactual binding of immunoglobulin reagents was encountered when goat antisera, with high levels of circulating immune complexes, formed the middle layer in the sandwich technique. This was attributed to the appearance of Fc receptors on the plasma membrane of degenerating acinar cells, and could be avoided by ultracentrifuging acinar cells, and could be avoided by ultracentrifuging the goat antisera prior to sue. In view of the fact that C3 cleavage represents an amplification loop in both the calssical and alternate pathways of complement activation, the lack of demonstrable C3 staining in tbe present experiments strongly suggests that complement plays no role in acinar cell necrosis in this model of pancreatitis.

Acute Disease↗

Expression of the IL-2 receptor on human renal proximal tubular epithelial cells.

The high-affinity receptor for interleukin (IL)-2, IL-2R, is composed of three chains, i.e., the alpha, beta, and gamma chains. Previous studies have shown that human proximal tubular epithelial cells (PTEC) express IL-2R alpha during inflammation, such as in the kidneys after renal transplantation or in crescentic glomerulonephritis. Furthermore, in this study it is shown that PTEC in culture produce more complement C3 after stimulation with IL-2, suggesting the presence of a functional IL-2R on PTEC. In these experiments, the binding of IL-2 to PTEC was analyzed. PTEC stimulated with IL-2 exhibited very low binding of digoxigenin-labeled IL-2; however, stimulation of PTEC with IL-2, in combination with interferon (IFN)-gamma, resulted in increased binding of the digoxigenin-labeled IL-2. In addition, it was found that IL-2, together with IFN-gamma, enhanced the production of C3 by PTEC from baseline levels of 69.6 +/- 3.4 to 198.6 +/- 3.3 ng of C3 per 10(6) cells. The surface expression of IL-2R alpha and IL-2Rbeta was analyzed using monoclonal antibodies. Although unstimulated PTEC hardly showed detectable expression of these chains, stimulation of PTEC with the combination of IL-2 and IFN-gamma resulted in increases of the index of mean fluorescence for the alpha- and beta-chains of 5.5 +/- 0.5 and 9.7 +/- 0.7, respectively. Reverse transcription-PCR analysis revealed mRNA expression not only for the alpha- and beta-chains, but also for the gamma-chain, after stimulation with the combination of IL-2 and IFN-gamma. This study demonstrates that PTEC express the high-affinity IL-2R and that the IL-2R might be involved in the regulation of production of complement component C3 found during renal inflammation.

Base Sequence↗

Human interleukin-6 acts as a co-factor for the up-regulation of C3 production by rat liver epithelial cells.

We investigated the role of human interleukin-6 (IL-6) in the regulation of the third component of complement (C3) biosynthesis by cultured rat liver epithelial cells. A natural human IL-6 (nh IL-6) preparation was shown to up-regulate C3 production, whereas an Escherichia coli-derived recombinant human IL-6 (rh IL-6) displayed no activity on C3 biosynthesis. However, the C3-stimulating activity of the nh IL-6 preparation was only partially reduced when treated with an antihuman IL-6 monoclonal antibody. Binding studies indicated that although it was devoid of any C3 stimulating activity, rh IL-6 specifically bound to hepatic cell receptors (Kd = 0.38 nM) and possessed the same binding affinity as nh IL-6. Furthermore, the substitution of natural IL-6 molecules for the recombinant IL-6 led to the recovery of the initial C3-stimulating activity. These studies demonstrated that human IL-6 alone does not stimulate rat liver epithelial cell C3 production but is able to accentuate the C3-stimulating activity of unrecognized components which are present in the nh IL-6 preparation. Human IL-6 thus appears to act as a co-factor for the up-regulation of hepatic C3 production.

Animals↗

Linkage heterogeneity between the C3 and LDLR and the APOA4 and APOA1 loci in baboons.

We detected linkage in baboons between loci for the third component of complement (C3) and the low-density lipoprotein receptor (LDLR), lod score = 5.53, and loci for apolipoprotein A1 (APOA1) and apolipoprotein AIV (APOA4), lod score = 14.59. We also found evidence for linkage heterogeneity among the baboon pedigrees between LDLR and C3 (P = 0.04) and between APOA1 and APOA4 (P = 0.01).

Animals↗

IgG and C3 receptors on polymorphonuclear leukocytes in phagocytosis. Role of C3-receptors in the attachment and ingestion phases.

The role of IgG and complement (C3) receptors in the adhesion and ingestion phases of immune complexes by normal human polymorphonuclear leukocytes (PMN) were examined. The immune complexes were sheep red cells (E) sensitized with IgG (EA) or IgM antibodies plus complement (EAC'). Both types of receptor sites are involved in the adhesion phase. Moreover IgG receptors are primarily involved in the ingestion phase. Nevertheless even C3-receptors may be sufficient for complete phagocytosis. Even if EAC' adhesion was still high, C3-receptor specific phagocytosis decreased parallel with the amount of the complement used for EA19S sensibilization. The role of receptor sites on human PMN in adhesion and ingestion phases is discussed.

Binding Sites, Antibody↗

HIV and HIV-infected cells differentially activate the human complement system independent of antibody.

The human retroviruses HTLV-I and HIV-I have previously been shown not to be lysed by human serum. An interaction between HIV and the complement system, however, has not been investigated in any detail. In this report we show that purified HIV as well as HIV-infected cells activate the complement system. In the case of virus-infected cells this activation is mediated by the alternative pathway of complement, whereas the classical pathway seems to be in operation for the triggering of the complement system by purified virus and recombinant envelope glycoprotein (gp 160). We demonstrate that this leads to the deposition of C3b and/or C3bi on the surface of infected cells. But the HIV-infected cells are not lysed by human complement. C3 fragments deposited on the surface of HIV-infected cells are capable of mediating immune adherence to complement receptor-bearing cells, such as human erythrocytes and phagocytes. Whether this might have an influence on infectivity of HIV for certain cells bearing complement receptors has yet to be shown.

Cell Line↗

Study of five haemogenetic markers (Gc, C3, Bf, Ag, and GALT) in six Indonesian populations and in 12 subgroups of Balinese.

In various ethnic groups of the Indonesian archipelago and of Bali, the polymorphisms of the serum proteins Gc globulin (vitamin D-binding protein), C3 (complement component 3), Bf (complement factor B), Ag x,y (lipoprotein allotypes), and of the red cell enzyme system GALT (galactose-1P-uridyltransferase) were analysed. Among the studied proteins, the Gc system was the most informative one for the anthropologist. Besides considerable differences of frequencies of the common alleles Gc*1F, Gc*1S and Gc*2, a number of rare alleles (1A1, 1A3, 1A8, 1A9, 1A12, 1C2, 1C21, 1C24, and 2C8) and some new ones (1C28, 1C29, 1C30, 2C9) were observed. The presence of Gc*1A1 demonstrates the relationship to the Australo-Melanesian populations, but Mongolian variants (1A3, 1A8, 1A9, 1C2) were also encountered. Within the C3 system a very high frequency of the C3*S allele was observed in all populations. The rare alleles C3*F0.55, C3S1, and C3*S0.5 were observed in some groups. A new allele (C3*F0.35) was detected in a Chinese individual and in a nobleman from Bali. The frequency of the Bf*F allele was rather low in general, and the Bf*S0.7 allele was found in three Indonesian individuals only. The Ag*(x) frequencies were rather high, as it is known for Asiatic populations. Variability among subgroups was not very pronounced. The GALT*2 allele (Duarte variant of the enzyme) was observed very rarely; however, it was present in several populations. Enzyme activities could not be determined, and therefore we cannot tell whether the galactosaemia gene (GALT*0) was present or not.

Complement C3↗

A 58-base-pair region of the human C3 gene confers synergistic inducibility by interleukin-1 and interleukin-6.

We have cloned the promoter for the human third component of complement (C3) gene and have identified sequences involved in its regulation during the acute-phase response. A construct linking 199 bp of the C3 promoter to the firefly luciferase gene was found to be very responsive to interleukin-1 (IL-1) and modestly responsive to interleukin-6 (IL-6) by transfection analysis in the human hepatoma line Hep3B2. Simultaneous treatment with the two cytokines showed a strong synergy between the actions of the two molecules. A 58-bp fragment (-127 to -70 bp) was shown by 5' and 3' deletional mutagenesis to contain cis-acting elements that mediated both the IL-1 response and the IL-1-plus-IL-6 synergistic response of this promoter. When coupled to a heterologous promoter, this fragment enabled the synergistic induction by IL-1 plus IL-6. Sequences homologous to the palindrome ACATTGCACAATCT, which mediates the induction of the IL-6 gene by IL-1 (S. Akira, H. Isshiki, T. Sugita, O. Tanabe, S. Kinoshita, Y. Nishio, T. Nakajima, T. Hirano, and T. Kishimoto, EMBO J. 9:1897-1906, 1990), and the core sequence of the IL-6-responsive element of the rat alpha 2-macroglobulin gene (CTGGGA; M. Hattori, L. J. Abraham, W. Northemann, and G. H. Fey, Proc. Natl. Acad. Sci. USA 87:2364-2368, 1990) are contained within this fragment in immediate juxtaposition and partially overlapping. Site-directed mutagenesis within this homology region drastically reduced the inducibility of the C3 promoter by either cytokine. DNase I footprinting analysis defined a binding site for the transcription factor CCAAT/enhancer-binding protein (C/EBP), which included the IL-1-responsive element-like sequence. No differences were seen between the footprints generated by using extracts from unstimulated and IL-1-stimulated Hep3B2 cells. However, gel retardation analyses revealed two IL-1-specific bands. The data suggest that the induction by IL-1 is mediated by a factor belonging to the family of C/EBP-related proteins.

Animals↗

Comparison of different assays for circulating immune complexes in age matched vasectomized and non-vasectomized men.

Sera from 46 men vasectomized for 5 yr and from 46 age-matched nonvasectomized men had previously been analyzed for circulating immune complexes (CICs) by four assays. Four additional CIC assays have now been performed on these sera: three Raji cell enzyme-linked immunosorbent assays (ELISAs) using alkaline phosphatase-conjugated antihuman IgG, IgA or C3 and the bull sperm ELISA. No significant differences in CIC levels were detected between the two groups using any of the assays. Results for two different sera obtained from 16 men 4 1/2 months apart correlated significantly for six of seven CIC assays evaluated in this way. In the bull sperm and Raji cell ELISAs, utilizing anti-IgG in the detection layer, the vasectomized men with sperm-agglutinating antibodies were found to have significantly higher CIC levels than those without sperm agglutinants. No association was found between the presence of sperm protamine antibody and levels of CICs. Since the vasectomized and control groups did not differ with respect to levels of CICs, immunoglobulins, or the complement C3 split product C3d, all 92 samples were combined into one group for further analysis. Serum IgG levels significantly correlated with the CIC levels in four of five CIC assays involving binding of IgG; IgA levels correlated with CIC determinations in the Raji-IgA assay, while plasma C3d levels correlated with the Raji-C3, the Raji-IgA, and the C1q-protein A binding assays. IgM levels did not correlate with activity in any assay. Finally, the degree of correlation between all eight CIC assays was determined, and significant positive correlations between assays were found in ten of twenty-eight comparisons.

Adult↗

C3 as substrate for adhesion of Streptococcus pneumoniae.

The ability of choline-binding protein A (CbpA) of Streptococcus pneumoniae to bind the third component of complement (C3) suggests possible interactions with opsonic C3 in the bloodstream or with C3 secreted by epithelial cells. The latter possibility was investigated by measuring C3 in supernatants of resting and cytokine-activated monolayers of type II pulmonary epithelial cells (A549 cells). Expression of C3 on the epithelial cell surface was confirmed by immunofluorescence. Epithelially produced C3 bound to CbpA, as determined by Western blot test. cbpa(-) mutants and lysates therefrom failed to bind C3, were completely deficient in adhesion to a matrix in which C3 was the sole substrate, and demonstrated a moderate yet significant decrease in adhesion to type II pulmonary epithelial cells. These results confirm the interaction of the pneumococcal protein CbpA and its substrate, C3, in 2 in vitro models of adhesion.

Bacterial Adhesion↗

Xenoantiserum to human C3 receptors: its preparation and effect on the C3b and C3d receptors of tonsil cells and the C3b receptors of erythrocytes and neutrophils.

Antisera directed against complement (C3) receptors on human tonsil cells were prepared and tested for their capacity to block specifically C3 receptors on various types of human cells. The antisera were capable of blocking both membrane-bound and solubilized C3 receptors of human tonsil cells. The C3b receptors of human erythrocytes and granulocytes were also blocked by the anti-C3 receptor sera. Sheep erythrocyte rosette formation was not affected. IgG-EoxA rosette formation was only slightly reduced by the anti-C3 receptor sera. Immunofluorescent staining with anti-C3 receptor sera revealed only a faint or negative staining of T cells and a distinct staining of EAC-reactive tonsil cells, lymphocytic leukaemia cells, and granulocytes. Absorption of the antisera with human serum proteins, brain, thymus, liver, EU-1 cell line cells, or trypsinized tonsil cells did not influence the capacity of the anti-C3 receptor sera to inhibit C3 receptors, whereas absorption with splenic tissue or tonsil cells completely removed the blocking activity of the anti-C3 receptor sera. Absorption with human erythrocytes or kidney removed only the inhibitory effect of the antisera on C3b receptors of tonsil cells, human erythrocytes, and granulocytes, but not on C3d receptors of tonsil cells. The results indicate that (a) the antisera prepared with the described procedure contained significant amounts of antibody against C3 receptors, (b) the receptors for C3b and C3d differe in antigenicity, and (c) the C3b receptors of tonsil cells, human erythrocytes, granulocytes, and probably glomerular cells have common antigenic sites.

Complement C3↗

[The influence of climatic treatment on the levels of complement and some lysosomal enzymes in serum of patients with asthma bronchiale (author's transl)].

It was found that the levels of CH50, C3, complement component. lysozyme, beta-D-glucuronidase, and acid phosphatase were increased in blood serum of patients with asthma bronchiale during a climatic treatment in the highmountain condition of Strbské Pleso (altitude 1,350 m). The increase of CH50 was monophasic, while the changes of other factors were biphasic--increasing at the beginning and falling down to the starting values at the end of the treatment. The changes observed are a part of the adaptation of organism to the high-mountain condition.

Acid Phosphatase↗