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Age-related macular degeneration (AMD) is a progressive chronic neurodegenerative retinal disease leading to vision loss, irreversible blindness, and visual impairment in older adults worldwide. Complement component 3 (C3) protein has been identified as the most predominant biomarker towards early diagnosis of AMD; therefore, there is an utmost requirement for non-invasive detection of C3 protein in the tear fluids of AMD patients. Considering this, we report an insightful electrochemical sensor capable of detecting clinically relevant concentrations ranging from 10 fg/mL to 1 μg/mL using electrospun indium-doped zinc oxide (InZnO) nanofibers as the transducing layer. The InZnO nanofibers have facilitated high anti-C3 antibody loading of 3.42 × 10−9 mol/cm2 and enhanced the overall charge transport mechanism at the sensor interface. The biofunctionalization process of the biosensor was investigated thoroughly using X-ray photoelectron spectroscopy (XPS) as well as different electrochemical techniques. The target C3 proteins were captured on the fabricated biosensor surface and determined through changes in charge transfer resistance (RCT) while executing electrochemical impedance spectroscopy (EIS) and peak current (Ip) in the case of cyclic voltammetry (CV) and differential pulse voltammetry (DPV), respectively. The InZnO nanofiber-based nano-biosensor demonstrated a very low limit of detections (LODs) of 5.214 fg/mL and 0.241 fg/mL with an excellent sensitivity of 4.6709 (ΔR/R) (g/mL)−1 cm−2 and 54.4939 (ΔIp/Ip)% (g/mL)−1 cm−2 for EIS and DPV techniques, respectively. By virtue of high antibody loading, ultrasensitive and ultra-selective capability, the indium-doped ZnO nanofibers show huge potential to be used as a high-performance diagnostic platform for AMD diagnosis. [ABSTRACT FROM AUTHOR]

Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid β protein precursor (APP) and neurotoxic amyloid β (Aβ) peptides in reactive astrocytes. Also, aberrant Ca2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer's disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aβ peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca2+ entry after complete Ca2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca2+ from the ER, preventing a rise in the resting cytosolic Ca2+ concentration. [ABSTRACT FROM AUTHOR]

IntroductionPlatelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy.MethodsIn this study, we investigated the role of complement in Escherichia coli (E. coli)-induced platelet aggregation in human whole blood, using Multiplate® aggregometry, flow cytometry, and confocal microscopy.Results and DiscussionWe found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli-induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli-induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli-induced platelet aggregation involved other blood cells. In conclusion, the E. coli-induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process.

Highly neurotoxic A1-reactive astrocytes have been associated with several human neurodegenerative diseases. Complement protein C3 expression is strongly upregulated in A1 astrocytes, and this protein has been shown to be a specific biomarker of these astrocytes. Several cytokines released in neurodegenerative diseases have been shown to upregulate the production of amyloid β protein precursor (APP) and neurotoxic amyloid β (Aβ) peptides in reactive astrocytes. Also, aberrant Ca2+ signals have been proposed as a hallmark of astrocyte functional remodeling in Alzheimer’s disease mouse models. In this work, we induced the generation of A1-like reactive astrocytes after the co-treatment of U251 human astroglioma cells with a cocktail of the cytokines TNF-α, IL1-α and C1q. These A1-like astrocytes show increased production of APP and Aβ peptides compared to untreated U251 cells. Additionally, A1-like astrocytes show a (75 ± 10)% decrease in the Ca2+ stored in the endoplasmic reticulum (ER), (85 ± 10)% attenuation of Ca2+ entry after complete Ca2+ depletion of the ER, and three-fold upregulation of plasma membrane calcium pump expression, with respect to non-treated Control astrocytes. These altered intracellular Ca2+ dynamics allow A1-like astrocytes to efficiently counterbalance the enhanced release of Ca2+ from the ER, preventing a rise in the resting cytosolic Ca2+ concentration.

Pseudomonas aeruginosa is a major cause of nosocomial bloodstream infections. This microorganism secretes two major proteases, alkaline protease A (AprA) and elastase B (LasB). Despite several in vitro studies having demonstrated that both purified proteases cleave a number of components of the immune system, their contribution to P. aeruginosa bloodstream infections in vivo remains poorly investigated. In this study, we used a set of isogenic mutants deficient in AprA, LasB or both to demonstrate that these exoproteases are sufficient to cleave the complement component C3, either soluble or deposited on the bacteria. Nonetheless, exoprotease-deficient mutants were as virulent as the wild-type strain in a murine model of systemic infection, in Caenorhabditis elegans and in Galleria mellonella. Consistently, the effect of the exoproteases on the opsonization of P. aeruginosa by C3 became evident four hours after the initial interaction of the complement with the microorganism and was not crucial to survival in blood. These results indicate that exoproteases AprA and LasB, although conferring the capacity to cleave C3, are not essential for the virulence of P. aeruginosa bloodstream infections. [ABSTRACT FROM AUTHOR]

Pseudomonas aeruginosa is a major cause of nosocomial bloodstream infections. This microorganism secretes two major proteases, alkaline protease A (AprA) and elastase B (LasB). Despite several in vitro studies having demonstrated that both purified proteases cleave a number of components of the immune system, their contribution to P. aeruginosa bloodstream infections in vivo remains poorly investigated. In this study, we used a set of isogenic mutants deficient in AprA, LasB or both to demonstrate that these exoproteases are sufficient to cleave the complement component C3, either soluble or deposited on the bacteria. Nonetheless, exoprotease-deficient mutants were as virulent as the wild-type strain in a murine model of systemic infection, in Caenorhabditis elegans and in Galleria mellonella. Consistently, the effect of the exoproteases on the opsonization of P. aeruginosa by C3 became evident four hours after the initial interaction of the complement with the microorganism and was not crucial to survival in blood. These results indicate that exoproteases AprA and LasB, although conferring the capacity to cleave C3, are not essential for the virulence of P. aeruginosa bloodstream infections.

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF, Bb is an enzyme that cleaves C3 and C5. However, CVF, Bb exhibits significantly different functional properties from C3b,Bb. Whereas both, CVF, Bb and C3b, Bb exhibit spontaneous decay-dissociation into the respective subunits, thereby eliminating the enzymatic activity, the CVF, Bb convertase is physico-chemically far more stable, decaying with a half-life that is more than two orders of magnitude slower than that of C3b,Bb. In addition, CVF, Bb is completely resistant to inactivation by Factors H and I. These two properties of CVF, Bb allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the physico-chemical stability of CVF,Bb, we have created recombinant hybrid proteins of CVF and human C3, based on structural differences between CVF and human C3b in the C-terminal C345C domain. Here we describe three human C3/CVF hybrid proteins which differ in only one, two, or five amino acid residues from earlier described hybrid proteins. In all three cases, the hybrid proteins containing CVF residues form more stable convertases, and exhibit stronger complement-depletion activity than hybrid proteins with human C3 residues. Three bonds between CVF residues and Factor Bb residues could be identified by crystallographic modeling that contribute to the greater stability of the convertases. • Hybrid proteins of human C3 and Cobra Venom Factor were recombinantly generated. • Point mutations allow the identification of important amino acids for convertase stability. • Three bonds between human Factor B and Cobra Venom Factor could be identified. [ABSTRACT FROM AUTHOR]

Abstract Background Sedentary behaviour, obesity and insulin resistance are associated with pro-inflammatory status. Limited data on whether physical activity modulates inflammatory status and counteracts obesity and insulin resistance associated low-grade inflammation exist. Our objective was to investigate associations between objectively measured physical activity and inflammatory status, and specifically whether substituting daily sedentary behaviour with light activity or moderate to vigorous physical activity (MVPA), is associated with beneficial alterations to the inflammatory profile among middle-aged adults and those at increased cardiometabolic risk (obese and insulin resistant subjects). Methods Data are from a sub-sample of the Mitchelstown cohort; a population-based cross-sectional sample of 2047 Irish adults. Physical activity intensity and duration were measured in 396 participants for 7-consecutive days using the GENEActiv accelerometer. Isotemporal regression analysis examined the associations between replacing 30 min per day of sedentary behaviour with equal amounts of light activity and MVPA on inflammatory factors (serum acute-phase reactants, adipocytokines, pro-inflammatory cytokines and white blood cells (WBC)). Results Reallocating 30 min of sedentary time with MVPA was associated with a more favourable inflammatory profile characterized by higher adiponectin and lower complement component C3 (C3), leptin, interleukin 6 (IL-6) and WBC concentrations (P 75th percentile) subjects. Among the non-obese and non-insulin resistant subjects substituting 30 min of sedentary behaviour with MVPA was associated with decreased C3, IL-6 and WBC concentrations. Conclusions Replacing sedentary behaviour with MVPA modulates pro-inflammatory status. These findings, which highlight the need for the developing randomized trials aimed at lowering cardiometabolic risk, warrant further investigation.

Abstract Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure. Most cases of aHUS are caused by uncontrolled complement activation due to genetic mutations in the alternative pathway of complement. More recently, mutations in the gene of coagulation system have also been identified in patients with aHUS. In Japan, the recent studies of aHUS have identified the unique genetic characteristics in our country and enabled us to revise the diagnostic criteria. In this article, we review the classification of TMAs and describe the pathophysiology, diagnosis, and management of aHUS. We also highlight current progress in clinical and basic research of the patients with aHUS in Japan.

Properdin (FP) is a positive regulator of the immune system stimulating the activity of the proteolytically active C3 convertase C3bBb in the alternative pathway of the complement system. Here we present two crystal structures of FP and two structures of convertase bound FP. A structural core formed by three thrombospondin repeats (TSRs) and a TB domain harbors the convertase binding site in FP that mainly interacts with C3b. Stabilization of the interaction between the C3b C-terminus and the MIDAS bound Mg2+ in the Bb protease by FP TSR5 is proposed to underlie FP convertase stabilization. Intermolecular contacts between FP and the convertase subunits suggested by the structure were confirmed by binding experiments. FP is shown to inhibit C3b degradation by FI due to a direct competition for a common binding site on C3b. FP oligomers are held together by two sets of intermolecular contacts, where the first is formed by the TB domain from one FP molecule and TSR4 from another. The second and largest interface is formed by TSR1 and TSR6 from the same two FP molecules. Flexibility at four hinges between thrombospondin repeats is suggested to enable the oligomeric, polydisperse, and extended architecture of FP. Our structures rationalize the effects of mutations associated with FP deficiencies and provide a structural basis for the analysis of FP function in convertases and its possible role in pattern recognition.

Complement component C3 is well recognized as the central mediator of complement system, whose activation is responsible for the immune surveillance and elimination of non-self-antigens. In this study, C3 gene (HcC3) from a pearl making mussel, Hyriopsis cumingii , was successfully identified. The putative HcC3 possessed the canonical domains and highly conserved functional residues of C3 family members. In phylogenetic analysis, HcC3 was also clustered into C3 subfamily and separated from α2 macroglobulin clade. HcC3 gene was constitutively expressed in a wide range of tissues of pearl mussels, among which the immune-related tissues like hemocytes got highest expression. After allograft surgery of mantle tissues for aquaculture pearl production, the gene expression of HcC3 exhibited a rapid upregulation on day 1, dropped back on day 3, peaked the value on day 7, and restored to the level similar to control samples on day 14 after mantle allograft. The biphasic expression within the two weeks post the surgery suggests the important roles for HcC3 in alloimmune responses and an intricate complement activation mechanism in mollusks during tissue allograft. • HcC3 was characterized as a typical member of C3 family with all the canonical features. • Phylogenetic analysis proved the gene divergence between C3 gene and A2M gene in pearl mussels. • Mantle allograft induced a rapid activation of C3. • HcC3 gene was in a biphasic expression in response to mantle transplantation. [ABSTRACT FROM AUTHOR]

Cobra venom factor (CVF) is the complement-activating protein in cobra venom. CVF is a structural and functional analog of complement component C3. CVF, like C3b, forms a convertase with factor B. This bimolecular complex CVF,Bb is an enzyme that cleaves C3 and C5. However, CVF,Bb exhibits significantly different functional properties from C3b,Bb. The CVF,Bb convertase is physico-chemically very stable, and completely resistant to an activation by Factors H and I. These two properties, in contrast to C3b,Bb, allow continuous activation of C3 and C5, and complement depletion in serum. In order to understand the structural basis for the functional differences between CVF and C3, we have created several hybrid proteins of CVF and human C3. Here we report that replacing the C-terminal 168 amino acid residues of human C3 with the corresponding residues from CVF results in a hybrid protein (HC3-1496) which is essentially a human C3 derivative exhibiting the functional properties of CVF. This result demonstrates that the important structures for the CVF-specific functions reside within the C-terminal 168 amino acid residues of CVF. We further demonstrate that reverting the 46 C-terminal CVF residues of HC3-1496 to human C3 sequence results in a hybrid protein (HC3-1496/1617) that exhibits a physico-chemically unstable convertase with only residual complement depleting activity. This result demonstrates that most, but not all, structural requirements for CVF activity reside within the 46 C-terminal amino acid residues. We also investigated the potential role of position 1633, which is an acidic residue in human C3 (glutamic acid) but a basic amino acid residue (histidine) in CVF. However, the charge at position 1633 appears to be of no functional relevance. Exchanging the neutral amino acids present in CVF at positions 1499 and 1501 with the two charged amino acids at these positions in human C3 (aspartic acid and lysine) resulted in a hybrid protein that exhibited significantly slower convertase formation although both binding to Factor B and C3 cleavage was not affected, demonstrating that the charged amino acid residues at these two positions interfere with the formation of the convertase. In conclusion, our work demonstrates that hybrid proteins of human C3 and CVF present valuable tools to identify functionally important amino acid residues in CVF. • We created hybrid proteins of human C3 and cobra venom factor (CVF) to identify structural requirements for CVF functions. • The 168 C-terminal amino acids are important for forming a stable convertase and conferring resistance to Factor H and I. • Most, but not all, of this activity is encoded in the 46 C-terminal amino acid residues. • The charge of the amino acid residue at position 1633 appears to be of no functional relevance. • The introduction of charged residues from human C3 at positions 1499 and 1501 resulted in slower convertase formation. [ABSTRACT FROM AUTHOR]

Properdin (FP) is a positive regulator of the immune system stimulating the activity of the proteolytically active C3 convertase C3bBb in the alternative pathway of the complement system. Here we present two crystal structures of FP and two structures of convertase bound FP. A structural core formed by three thrombospondin repeats (TSRs) and a TB domain harbors the convertase binding site in FP that mainly interacts with C3b. Stabilization of the interaction between the C3b C-terminus and the MIDAS bound Mg2+ in the Bb protease by FP TSR5 is proposed to underlie FP convertase stabilization. Intermolecular contacts between FP and the convertase subunits suggested by the structure were confirmed by binding experiments. FP is shown to inhibit C3b degradation by FI due to a direct competition for a common binding site on C3b. FP oligomers are held together by two sets of intermolecular contacts, where the first is formed by the TB domain from one FP molecule and TSR4 from another. The second and largest interface is formed by TSR1 and TSR6 from the same two FP molecules. Flexibility at four hinges between thrombospondin repeats is suggested to enable the oligomeric, polydisperse, and extended architecture of FP. Our structures rationalize the effects of mutations associated with FP deficiencies and provide a structural basis for the analysis of FP function in convertases and its possible role in pattern recognition. [ABSTRACT FROM AUTHOR]

Largemouth bronze gudgeon (Coreius guichenoti) is a fish endemic to the middle and upstream reaches of the Yangtze River in China. Indoor culture and domestication of C. guichenoti have yet to see success in part because of Ichthyophthirius multifiliis infection, which causes 100% mortality in these fish. Here, we investigated whether the complement components C3 and C9 in C. guichenoti play a role in immune reactions against I. multifiliis. Full-length C3 and C9 cDNA sequences were determined and analyzed. The C3 cDNA sequence comprised 5142 bp encoding 1643 amino acids. The C9 cDNA sequence comprised 2206 bp with an open reading frame of 2085 bp. The mRNA expression of C3 and C9 genes was detected in the liver, spleen, and trunk kidney, and as well as that of C3 gene in head kidney. I. multifiliis infection significantly upregulated mRNA expression of C3 and C9 in liver and spleen. However, no transcript was detected in the skin of C. guichenoti post-infection with I. multifiliis , and might be responsible for C. guichenoti to be vulnerable to the parasite. The results suggest that C3 and C9 are involved in defense against the ectoparasite I. multifiliis , and promote understanding function of the complement components C3 and C9 in C. guichenoti. • Full-length C3 and C9 cDNA sequences from largemouth bronze gudgeon (Coreius guichenoti) were cloned and analyzed. • The tissue expression patterns of C3 and C9 in C. guichenoti were measured with in situ hybridization and RT-PCR techniques. • The transcripts of C3 and C9 genes in response to Ichthyophthirius multifiliis infection were studied with RT-PCR technique. [ABSTRACT FROM AUTHOR]

Abstract The complement system plays an important role in protecting fish against attack by pathogens early in life. Complement component C3 is a central component in the complement system. The present work aimed to clone the full length C3 cDNA sequence of southern catfish (Silurus meridionalis), detect the tissue expression patterns of C3, investigate the ontogeny of C3 in embryo and larva, and assess the expression of C3 in response to pathogen infection. The full length C3 cDNA sequence of 5157 bp with an open reading frame (ORF) of 4938 bp was cloned from southern catfish. The deduced amino acid sequence showed similarity with other teleost fish. The mRNA expression of C3 was detected in liver, spleen, stomach, intestine, and head kidney with RT-PCR and in situ hybridization. Whole mount in situ hybridization results revealed that C3 was first expressed in the yolk syncytial layer at 34 h post fertilization (hpf), followed by the liver at 36 h post hatching (hph). When challenged with Aeromonas hydrophila , the transcripts of C3 showed a significant up-regulation in liver and spleen at 24 h. The results suggested that complement C3 played a key role in defense against invading pathogens in the early development stages of southern catfish. Therefore, these results provide important information to understand the functions of C3 during fish early development in Siluriformes. Highlights • The full length complement C3 cDNA sequence was identified and analyzed from southern catfish (Silurus meridionalis). • The ontogeny of C3 in southern catfish was investigated with whole mount in situ hybridization and RT-PCR techniques. • The mRNA of C3 gene in tissues of southern catfish infected with Aeromonas hydrophila was studied with RT-PCR technique. [ABSTRACT FROM AUTHOR]

von Willebrand factor (VWF) is a multimeric protein composed of monomeric subunits (∼280 kD) linked by disulfide bonds. During hemostasis and thrombosis, ultralarge (UL) VWF (ULVWF) multimers initiate platelet adhesion. In vitro, human C3 binds to ULVWF multimeric strings secreted by and anchored to human endothelial cell to promote the assembly and activation of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b) of the alternative complement pathway (AP). The purified and soluble C3 avidly binds to recombinant human VWF A1A2A3, as well as the recombinant isolated human VWF A3 domain. Notably, the binding of soluble human ULVWF multimers to purified human C3 was blocked by addition of a monovalent Fab fragment antibody to the VWF A3 domain. We conclude that the A3 domain in VWF/ULVWF contains a docking site for C3. In contrast, purified human C4, an essential component of the classical and lectin complement pathways, binds to soluble, isolated A1, but not to ULVWF strings secreted by and anchored to endothelial cells. Our findings should facilitate the design of new therapeutic agents to suppress the initiation of the AP on ULVWF multimeric strings during thrombotic and inflammatory disorders.

Osteogenesis imperfecta (OI) is a disease characterized by low bone mass and bony fragility. This study investigated the serum proteomic profiles and their correlation with bone density for OI cases. Methods: Twenty OI patients and 20 control participants were included. Comparative serum proteomic profiles were analyzed by two-dimensional electrophoresis and tandem mass spectrometry. Serum protein levels were measured by enzyme-linked immunosorbent assay. Cutoff values and areas under the curve were estimated by the receiver operating characteristic curve. Bone mineral density data was obtained from all OI patients. Results: Candidate proteins identified by electrophoresis were complement component C3 (C3), vitamin D-binding protein (DBP), and haptoglobin (HP). Enzyme-linked immunosorbent assay validation showed that OI patients had decreased C3 and DBP and increased HP. The results were not affected by age or bisphosphonate use. Serum C3 levels significantly correlated with bone mineral density of the lumbar spine and hip. C3 had the greatest areas under the curve to distinguish OI from healthy controls. Conclusion: Serum C3, DBP, and HP are emerging serologic signatures for OI. Concentrations of serum C3 correlated with the T score of OI patients. C3 had the greatest areas under the curve of the three proteins to distinguish OI from healthy controls.

Abstract: Control of mycobacterial infection constitutes a priority for human and animal health worldwide. However, effective vaccines are needed for the control of human and animal tuberculosis (TB). Adult zebrafish have become a useful model for studying the pathophysiology of mycobacterial infection and for the development of novel interventions for TB control and prevention. Recently, parenteral and oral immunization with the heat‐inactivated Mycobacterium bovis vaccine (M. bovis IV) protected wild boar against TB. The objectives of this study were to provide additional support for the role of M. bovis IV in TB control using the zebrafish model and to conduct the first trial with this vaccine for the control of fish mycobacteriosis. The results showed that M. bovis IV protected zebrafish against mycobacteriosis caused by low and high infection doses of Mycobacterium marinum and provided evidence suggesting that the protective mechanism elicited by M. bovis IV in zebrafish as in other species is based on the activation of the innate immune response through the C3 pathway, with a role for the regulatory protein Akr2 in this process. These results encourage the use of M. bovis IV for TB control in different species. [ABSTRACT FROM AUTHOR]

Background: Sedentary behaviour, obesity and insulin resistance are associated with pro-inflammatory status. Limited data on whether physical activity modulates inflammatory status and counteracts obesity and insulin resistance associated low-grade inflammation exist. Our objective was to investigate associations between objectively measured physical activity and inflammatory status, and specifically whether substituting daily sedentary behaviour with light activity or moderate to vigorous physical activity (MVPA), is associated with beneficial alterations to the inflammatory profile among middle-aged adults and those at increased cardiometabolic risk (obese and insulin resistant subjects). Methods: Data are from a sub-sample of the Mitchelstown cohort; a population-based cross-sectional sample of 2047 Irish adults. Physical activity intensity and duration were measured in 396 participants for 7-consecutive days using the GENEActiv accelerometer. Isotemporal regression analysis examined the associations between replacing 30 min per day of sedentary behaviour with equal amounts of light activity and MVPA on inflammatory factors (serum acute-phase reactants, adipocytokines, pro-inflammatory cytokines and white blood cells (WBC)). Results: Reallocating 30 min of sedentary time with MVPA was associated with a more favourable inflammatory profile characterized by higher adiponectin and lower complement component C3 (C3), leptin, interleukin 6 (IL-6) and WBC concentrations (P < 0.05). No significant effects were noted with substitution of sedentary time with light activity. Among the obese subjects replacing sedentary behaviour with an equivalent amount of MVPA was associated with lower WBC counts (P < 0.05); no associations were detected among the insulin resistant (HOMA-IR >75th percentile) subjects. Among the non-obese and non-insulin resistant subjects substituting 30 min of sedentary behaviour with MVPA was associated with decreased C3, IL-6 and WBC concentrations. Conclusions: Replacing sedentary behaviour with MVPA modulates pro-inflammatory status. These findings, which highlighttheneedforthedevelopingrandomizedtrialsaimedatlowering cardiometabolic risk, warrant further investigation. [ABSTRACT FROM AUTHOR]

Mastitis is an infectious disease of the mammary gland that leads to reduced milk production and change in milk composition. Complement component C3 plays a major role as a central molecule of the complement cascade involving in killing of microorganisms, either directly or in cooperation with phagocytic cells. C3 cDNA were isolated, from Egyptian buffalo and cattle, sequenced and characterized. The C3 cDNA sequences of buffalo and cattle consist of 5025 and 5019 bp, respectively. Buffalo and cattle C3 cDNAs share 99% of sequence identity with each other. The 4986 bp open reading frame in buffalo encodes a putative protein of 1661 amino acids-as in cattle-and includes all the functional domains. Further, analysis of the C3 cDNA sequences detected six novel single-nucleotide polymorphisms (SNPs) in buffalo and three novel SNPs in cattle. The association analysis of the detected SNPs with milk somatic cell score as an indicator of mastitis revealed that the most significant association in buffalo was found in the C >A substitution (ss: 1752816097) in exon 27, whereas in cattle it was in the C >T substitution (ss: 1752816085) in exon 12. Our findings provide preliminary information about the contribution of C3 polymorphisms to mastitis resistance in buffalo and cattle. [ABSTRACT FROM AUTHOR]

Background: Abnormal activation of the complement system contributes to some central nervous system diseases but the role of complement in HIV-associated neurocognitive disorder (HAND) is unclear.Methods: We used real-time PCR and immunohistochemistry to detect complement expression in postmortem brain tissue from HAND patients and controls. To further investigate the basis for viral induction of gene expression in the brain, we studied the effect of HIV on C3 expression by astrocytes, innate immune effector cells, and targets of HIV. Human fetal astrocytes (HFA) were infected with HIV in culture and cellular pathways and factors involved in signaling to C3 expression were elucidated using pharmacological pathway inhibitors, antisense RNA, promoter mutational analysis, and fluorescence microscopy.Results: We found significantly increased expression of complement components including C3 in brain tissues from patients with HAND and C3 was identified by immunocytochemistry in astrocytes and neurons. Exposure of HFA to HIV in culture-induced C3 promoter activity, mRNA expression, and protein production. Use of pharmacological inhibitors indicated that induction of C3 expression by HIV requires NF-κB and protein kinase signaling. The relevance of NF-κB regulation to C3 induction was confirmed through detection of NF-κB translocation into nuclei and inhibition through overexpression of the physiological NF-κB inhibitor, I-κBα. C3 promoter mutation analysis revealed that the NF-κB and SP binding sites are dispensable for the induction by HIV, while the proximal IL-1β/IL-6 responsive element is essential. HIV-treated HFA secreted IL-6, exogenous IL-6 activated the C3 promoter, and anti-IL-6 antibodies blocked HIV activation of the C3 promoter. The activation of IL-6 transcription by HIV was dependent upon an NF-κB element within the IL-6 promoter.Conclusions: These results suggest that HIV activates C3 expression in primary astrocytes indirectly, through NF-κB-dependent induction of IL-6, which in turn activates the C3 promoter. HIV induction of C3 and IL-6 in astrocytes may contribute to HIV-mediated inflammation in the brain and cognitive dysfunction. [ABSTRACT FROM AUTHOR]

Complement component 3 (C3) is one of the proteins associated with complement cascades. C3 plays an essential role in three different pathways-the alternative, classical, and lectin pathways. It is well known that cytokines activate complement system and increase complement component C3 production. In the current study, we found that lipoteichoic acid isolated from Lactobacillus plantarum K8 (pLTA) inhibited tumor necrosis factor-alpha (TNF-α)- or interferon-gamma (IFN-γ)-mediated C3 mRNA and protein expression in HaCaT cells. pLTA inhibited C3 expression through the inhibition of the phosphorylation of p65 and p38 in the TNF-α treated cells, while the inhibition of STAT1/2 and JAK2 phosphorylation by pLTA contributed to the reduction of C3 in IFN-γ-treated cells. When mice were pre-injected with pLTA followed by re-injection of TNF-α, serum C3 level was decreased as compared to TNF-α-injected only. Further studies revealed that membrane attack complex (MAC) increased by TNF-α injection was lessened in pLTA-pre-injected mice. A bactericidal assay using mouse sera showed that MAC activity in pLTA-pre-injected mice was lower than in TNF-α only-injected mice. These results suggest that pLTA can suppress inflammatory cytokine-mediated complement activation through the inhibition of C3 synthesis. pLTA application has the potential to alleviate complement-mediated diseases caused by excessive inflammation. [ABSTRACT FROM AUTHOR]

Deposition of extra cellular material is a feature of retinal ageing resulting in reduced outer retinal perfusion that can be associated with disease. This has been documented for Bruch's membrane (BM) and is elevated in mice that are complement factor H knock outs (Cfh-/-) and proposed as a model for macular degeneration. Here we ask if aged deposition occurs to the outer retinal blood supply before transmission across BM in 12 month old C57Bl and Cfh-/- mice and if differences exist at this level between these mice. We show arterial deposition of age related proteins complement component C3 with immunostaining and Western blot. Results showed deposition of C3 in all internal lamina of choroid blood vessels and three fragments were revealed from western blot which decrease significantly in Cfh-/- mice choroid. [ABSTRACT FROM AUTHOR]

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, β-glucan (MacroGard ® ) in turbot ( Scophthalmus maximus ). Rotifers ( Brachionus plicatilis ) were incubated with or without yeast β-1,3/1,6-glucan in form of MacroGard ® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of β-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard ® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by β-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70 , tnf-α and il-1β was observed. We conclude that the administration of MacroGard ® induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot. [ABSTRACT FROM AUTHOR]

The sub-clinical form of mastitis is difficult to detect and causes huge economic loss to the dairy industry. It has become a threat to public health at large, thus there is a need for definite diagnosis of the disease. Therefore, this study was undertaken to identify the novel diagnostic marker for the detection of the sub-clinical form of mastitis. Two-dimensional gel analysis of the whey protein fraction of normal and mastitis milk samples revealed the presence of proteose peptone component 3 precursor, Trypsin precursor, complement component-C3, Ig heavy chain precursors and a C-type lectin domain as differentially expressed protein during the early stage of mastitis. Of these proteins identified, complement component-C3 was tested for its diagnostic potential. Western blot analysis of the milk whey of sub-clinical mastitis cases (M+, M++ & M+++) identified the accumulation of C3a, an activated product of complement component-C3. Further, the hemolytic activity of the above milk whey samples positively correlated with the somatic cell count. As C3a is already reported as an anaphylotoxic agent, it chemo tactically attracts lymphocytes at the site of inflammation, the detection of which in the milk whey can be of diagnostic importance for sub-clinical mastitis.

Background/purpose: Osteogenesis imperfecta (OI) is a disease characterized by low bone mass and bony fragility. This study investigated the serum proteomic profiles and their correlation with bone density for OI cases.Methods: Twenty OI patients and 20 control participants were included. Comparative serum proteomic profiles were analyzed by two-dimensional electrophoresis and tandem mass spectrometry. Serum protein levels were measured by enzyme-linked immunosorbent assay. Cutoff values and areas under the curve were estimated by the receiver operating characteristic curve. Bone mineral density data was obtained from all OI patients.Results: Candidate proteins identified by electrophoresis were complement component C3 (C3), vitamin D-binding protein (DBP), and haptoglobin (HP). Enzyme-linked immunosorbent assay validation showed that OI patients had decreased C3 and DBP and increased HP. The results were not affected by age or bisphosphonate use. Serum C3 levels significantly correlated with bone mineral density of the lumbar spine and hip. C3 had the greatest areas under the curve to distinguish OI from healthy controls.Conclusion: Serum C3, DBP, and HP are emerging serologic signatures for OI. Concentrations of serum C3 correlated with the T score of OI patients. C3 had the greatest areas under the curve of the three proteins to distinguish OI from healthy controls. [ABSTRACT FROM AUTHOR]

Introduction: Platelets have essential functions as first responders in the immune response to pathogens. Activation and aggregation of platelets in bacterial infections can lead to life-threatening conditions such as arterial thromboembolism or sepsis-associated coagulopathy., Methods: In this study, we investigated the role of complement in Escherichia coli ( E. coli) -induced platelet aggregation in human whole blood, using Multiplate ® aggregometry, flow cytometry, and confocal microscopy., Results and Discussion: We found that compstatin, which inhibits the cleavage of complement component C3 to its components C3a and C3b, reduced the E. coli -induced platelet aggregation by 42%-76% (p = 0.0417). This C3-dependent aggregation was not C3a-mediated as neither inhibition of C3a using a blocking antibody or a C3a receptor antagonist, nor the addition of purified C3a had any effects. In contrast, a C3b-blocking antibody significantly reduced the E. coli -induced platelet aggregation by 67% (p = 0.0133). We could not detect opsonized C3b on platelets, indicating that the effect of C3 was not dependent on C3b-fragment deposition on platelets. Indeed, inhibition of glycoprotein IIb/IIIa (GPIIb/IIIa) and complement receptor 1 (CR1) showed that these receptors were involved in platelet aggregation. Furthermore, aggregation was more pronounced in hirudin whole blood than in hirudin platelet-rich plasma, indicating that E. coli -induced platelet aggregation involved other blood cells. In conclusion, the E. coli -induced platelet aggregation in human whole blood is partly C3b-dependent, and GPIIb/IIIa and CR1 are also involved in this process., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Landsem, Emblem, Lau, Christiansen, Gerogianni, Karlsen, Mollnes, Nilsson and Brekke.)

Activation of the complement cascade (CC) with myocardial infarction (MI) acutely initiates immune cell infiltration, membrane attack complex formation on injured myocytes, and exacerbates myocardial injury. Recent studies implicate the CC in mobilization of stem/progenitor cells and tissue regeneration. Its role in chronic MI is unknown. Here, we consider complement component C3, in the chronic response to MI. C3 knockout (KO) mice were studied after permanent coronary artery ligation. C3 deficiency exacerbated myocardial dysfunction 28 days after MI compared to WT with further impaired systolic function and LV dilation despite similar infarct size 24 hours post-MI. Morphometric analysis 28 days post-MI showed C3 KO mice had more scar tissue with less viable myocardium within the infarct zone which correlated with decreased c-kitpos cardiac stem/progenitor cells (CPSC), decreased proliferating Ki67pos CSPCs and decreased formation of new BrdUpos/α-sarcomeric actinpos myocytes, and increased apoptosis compared to WT. Decreased CSPCs and increased apoptosis were evident 7 days post-MI in C3 KO hearts. The inflammatory response with MI was attenuated in the C3 KO and was accompanied by attenuated hematopoietic, pluripotent, and cardiac stem/progenitor cell mobilization into the peripheral blood 72 hours post-MI. These results are the first to demonstrate that CC, through C3, contributes to myocardial preservation and regeneration in response to chronic MI. Responses in the C3 KO infer that C3 activation in response to MI expands the resident CSPC population, increases new myocyte formation, increases and preserves myocardium, inflammatory response, and bone marrow stem/progenitor cell mobilization to preserve myocardial function. S tem C ells 2014;32:2502-2515 [ABSTRACT FROM AUTHOR]

Lung cancer is the leading cause of cancer-related mortality worldwide. The complement component 3 (C3) is a central protein of the complement system, expressed in numerous cancer tissues and considered crucial for tumor progression. This study aimed to investigate the prognostic value of C3 in non-small cell lung cancer (NSCLC) and the underlying mechanisms. We used immunohistochemistry to observe the expression of C3 in malignant pulmonary lesion specimens from biopsy of 80 patients with NSCLC at stages I-III, who underwent lobectomy. We further assessed the correlation between C3 expression and a number of clinical features, as well as its prognostic value. To investigate the mechanism by which C3 exerts its effects, the correlation of C3 expression to T lymphocyte infiltration was also assessed. There was no significant correlation between the C3 level and clinical features such as gender, smoking status, degree of differentiation, histological type and malignant tumor stage based on the TNM classification system, while a significant correlation was found to age. However, analysis of overall survival (OS) and disease-free survival (DFS) rates showed that low C3 expression was related to poor prognosis. Univariate survival analysis revealed that C3 level and TNM stage are independent prognostic factors. Multivariate analysis demonstrated that the low level of C3 and TNM stage are also associated with poor prognosis. Furthermore, in tissues from biopsies, the C3 level positively correlated to the number of infiltrated CD4+ and CD8+ T lymphocytes (P<0.01). These findings indicate that C3 is a valuable marker for prognostic evaluation of NSCLC and may be considered as a therapeutic target for the treatment of lung cancer. [ABSTRACT FROM AUTHOR]

Abstract: Complement component C3 is a key protein in the complement system whose activation is essential for all the important functions performed by this system. In this study, the complete C3 cDNA sequence was isolated from the miiuy croaker (Miichthys miiuy), which was high similarity to other complement C3. In this study, we report the primary sequence, the tissue expression profile, the polypeptide domain architecture and the phylogenetic analysis of miiuy croaker C3 gene. Rapid amplification of the cDNA ends (RACE) yielded the full open reading frame of this protein (4974 bp), and subsequent analysis indicated that the M. miiuy C3 gene encoded a protein of 1657 amino acids. The deduced amino acid sequence showed that M. miiuy C3 has conserved residues and domains known to be critical for C3 function. Phylogenetic analysis showed that miiuy croaker was most closely related to Epinephelus coioides. Expression analysis showed that C3 was expressed differentially in miiuy croaker tissues, while liver was the main source of C3 expression. Infection of miiuy croaker with Vibrio anguillarum resulted in significant changes expression of C3 gene in the immune-related tissues. These results showed that C3 gene might play an important role in immune mechanisms. [Copyright &y& Elsevier]

Cobra Venom Factor (CVF) is the complement-activating protein in cobra venom. CVF is structurally and functionally highly homologous to complement component C3. CVF, like C3b, the activated form of C3, forms a bimolecular complex with Factor B in serum, called C3/C5 convertase, an enzyme which activates complement components C3 and C5. Despite the high degree of homology, the two C3/C5 convertases exhibit significant functional differences. The most important difference is that the convertase formed with CVF (CVF,Bb) is physico-chemically far more stable than the convertase formed with C3b (C3b,Bb). In addition, the CVF,Bb convertase and CVF are completely resistant to inactivation by the complement regulatory proteins Factor H and Factor I. Furthermore, the CVF,Bb enzyme shows efficient C5-cleaving activity in fluid phase. In contrast, the C3b,Bb enzyme is essentially devoid of fluid-phase C5-cleaving activity. By taking advantage of the high degree of sequence identity at both the amino acid (85%) and DNA levels (93%) between CVF and cobra C3, we created hybrid proteins of CVF and cobra C3 where sections, or only a few amino acids, of the CVF sequence were replaced with the homologous amino acid sequence of cobra C3. In a first set of experiments, we created five hybrid proteins, termed H1 through H5, where the cobra C3 substitutions collectively spanned the entire length of the CVF protein. We also created three additional hybrid proteins where only four or five amino acid residues in CVF were exchanged with the corresponding amino acid residues from cobra C3. Collectively, these hybrid proteins, representing loss-of-function mutants of CVF, allowed the identification of regions and individual amino acid residues important for the CVF-specific functions. The results include the observation that the CVF β-chain is crucially important for forming a stable convertase, whereas the CVF α-chain appears to harbor no CVF-specific functions. Furthermore, the CVF γ-chain is additionally important for the fluid-phase C5-cleaving activity of CVF,Bb. Interestingly, the structural changes in the individual hybrid proteins differentially affected the molecular functions of the CVF,Bb enzyme such as convertase formation, C3 cleavage, and C5 cleavage. [ABSTRACT FROM AUTHOR]

ABSTRACT: Aim: We investigated efficacy and therapeutic mechanisms of tonsillectomy for intractable childhood IgA nephropathy. Five patients refused tonsillectomy. Among 25 patients, 19 patients were able to evaluate histological findings before and after surgery. Patients with poor ( n = 7) or relatively poor ( n = 18) histologically determined prognosis and an age of at least 7 years, together with proteinuria of at least 0.3 g/day or severe persisting despite ongoing drug treatment, are candidates for surgery. Patients were grouped by interval between diagnosis of IgA nephropathy and tonsillectomy (within 3 years; early group vs 3 years or later; later group). Patients underwent kidney biopsy shortly before and 1 to 2 years after tonsillectomy. Results: Proteinuria was reduced after tonsillectomy over 2 years of follow-up in both early and later groups compared with proteinuria in the 6 months preceding surgery. Complete remission was achieved in 10 patients, most often among those having surgery within 3 years, while patients refusing surgery failed to attain complete remission of urinary findings. Histological activity decreased in both groups, significantly when surgery was early. Complement component C3 deposition and activated macrophages in glomeruli decreased after tonsillectomy, especially with early surgery. Conclusion: Tonsillectomy improved clinicopathological features in relatively severe paediatric IgA nephropathy, especially with the early-surgery group. Therapeutic mechanisms may include inhibition of complement activity in glomeruli and glomerular infiltration by activated macrophages. [ABSTRACT FROM AUTHOR]

Abstract: Complement component C3 is a key molecule in the complement system whose activation is essential for all the important functions performed by this system. In this study, we examined a temperature-subtracted library from the orange-spotted grouper (Epinephelus coioides), and identified a sequence with high similarity to other complement C3 proteins. Rapid amplification of the cDNA ends (RACE) yielded the full open reading frame of this protein, and subsequent analysis indicated that the Ec-C3 (E. coioides-C3) gene encodes a protein of 1657 amino acid residues with a molecular mass of 184.56 kDa. The deduced amino acid sequence showed that Ec-C3 has conserved residues and domains known to be critical for C3 function. RT-PCR assays showed that under normal physiological conditions Ec-C3 mRNA is most strongly expressed in the liver, but relatively high levels were also found in many other tissues, including intestine, muscles, gills, hemocytes, heart, spleen, head, kidney and brain. Further analysis of Ec-C3 gene expression in liver tissue by quantitative real-time PCR demonstrated that Ec-C3 transcript levels increased when the fish were exposed to both pH and temperature stress, but the time when its expression level peaked differed under these stresses. The results show that Ec-C3 mRNA expression in the orange-spotted grouper is influenced by pH and temperature stress and that Ec-C3 may play an important role in antioxidation mechanisms. [Copyright &y& Elsevier]

A fragment of a homolog of the complement component C3 gene was cloned and sequenced from the starfish Asterias rubens. A phylogenetic analysis of the gene, termed ArC3-like, demonstrated its close similarity to the C3 gene homolog of deuterostome invertebrates. A high level of ArC3-like mRNA expression was observed in circulating cells (coelomocytes), a gut derivate (hepatopancreas), and the male gonad, but not in the stomach, female gonad, and rectal gland of A. rubens. ArC3-like gene expression was detected in all types of starfish coelomocytes, including lymphocyte-like cells and granular and nongranular amebocytes. Bacterial lipopolysaccharide (LPS) injected into the coelomic cavity of starfish increased the ArC3-like gene expression in coelomocytes and the hepatopancreas as compared to the control level (sterile sea water injection). The level of ArC3-like gene expression in coelomocytes in response to LPS reached its maximum 6 h after the injection and decreased to the baseline level 24 h after the injection. In the hepatopancreas, the level of ArC3-like gene expression reached its maximum 6–12 h after the LPS injection stimulation and remained high even 48 h after the injection. A long-term upregulation in response to LPS was demonstrated for the ArC3-like gene. [ABSTRACT FROM AUTHOR]

Abstract: Cobra venom factor (CVF) is a structural and functional analog of complement C3 isolated from cobra venom. Both CVF and C3b can bind factor B and subsequently form the bimolecular C3/C5 convertases CVF,Bb or C3b,Bb, respectively. The two homologous enzymes exhibit several differences of which the difference in physico-chemical stability is most important, allowing continuous activation of C3 and C5 by CVF,Bb, leading to serum complement depletion. Here we describe the detailed functional properties of two hybrid proteins in which the 113 or 315 C-terminal residues of C3 were replaced with corresponding CVF sequences. Both hybrid proteins formed stable convertases that exhibited C3-cleaving activity, although at different rates. Neither convertase cleaved C5. Both convertases showed partial resistance to inactivation by factors H and I, allowing them to deplete complement in human serum. These data demonstrate that functionally important structural differences between CVF and C3 are located in the very C-terminal region of both homologous proteins, and that small substitutions in human C3 with homologous CVF sequence result in C3 derivatives with CVF-like functions. Such hybrid proteins are important tools to study the structure/function relationships in both C3 and CVF, and these “humanized CVF” proteins may become reagents for therapeutic complement depletion. [Copyright &y& Elsevier]

We have studied the impact of complement component 3 (C3) deficiency on the progression of lethal Babesia rodhaini infection in immune mice. A B. gibsoni ribosomal phosphoprotein P0 (BgP0) previously reported to be a cross-protective antigen against Babesia infection was used to immunize C57BL/6 wild-type (WT) and C3-deficient (C3–/–) mice. Test mice were immunized intraperitoneally (i.p.) with recombinant BgP0 (rBgP0), while controls either were immunized with PBS or did not receive any immunization. Following the immunization regime, test WT mice induced a specifically strong humoral response consisting of mixed immunoglobulins IgG1 and IgG2 associated with high production of IFN-γ in the supernatant of splenocytes. While test C3–/– mice had significantly decreased total IgG, IgG1 and IgG2b responses, the secretions of IL-12 and IFN-γ tended to be lower than those in WT mice. Furthermore, partial protection was only observed in rBgP0-immunized WT mice but not in C3–/– mice or controls. Indeed, rBgP0-immunized WT mice showed significant reductions in the initiation of parasitaemia correlated with delayed mortalities and considerable survival rates. Taken together, our results indicate that cross-protection was impaired in C3–/– mice in view of the decrease in the antibody responses and cytokine production and the high susceptibility to infection. [ABSTRACT FROM AUTHOR]

Abstract: Fish embryos and hatchlings are exposed to pathogens long before maturation of their lymphoid organs. Little is known about defence mechanisms during the earliest stages of life, but innate mechanisms may be essential for survival. The complement system in fish is well developed and represents a major part of innate immunity. Complement factor 3 (C3) is central subsequent to activation of all pathways of the complement system, leading to inflammatory reactions, such as chemotaxis, opsonisation and lysis of pathogens. Hepatocytes represent the major source of C3, but modern molecular biological methods have confirmed that C3 is synthesised at multiple sites. Our main objective was to study the ontogeny of C3 in Atlantic salmon by mapping the commencement of synthesis and localisation of proteins. Eggs, embryos, hatchlings and adult fish were analysed for the presence of C3 mRNA and proteins. From immunohistochemical studies, C3 proteins were detected at several extrahepatic sites, such as the skeletal muscle, developing notochord and chondrocytes of the gill arch. Immunoblotting revealed presence of C3 proteins in the unfertilised egg, but C3 mRNA was only detected after fertilisation by real-time RT-PCR. Taken together, the results implicated the maternal transfer of C3 proteins as well as novel non-immunological functions during development. [Copyright &y& Elsevier]

Abstract: Sjögren''s syndrome (SjS) is a human autoimmune disease characterized by the loss of exocrine function as a result of a chronic immune attack directed primarily against the salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). NOD.B10-H2 b mice manifest many features of SjS, exhibiting exocrine gland dysfunction concomitant with leukocyte infiltration of the salivary and lacrimal glands. Recent studies have shown that both SjS patients and NOD.B10-H2 b mice exhibit increased B lymphocyte survival, B cell hyper-reactivity and hyper-gammaglobulinemia with high production of autoantibodies. To study the possible influence of complement on the development and expression of SjS-like disease of the NOD.B10-H2 b , we have utilized a prophylactic treatment with CVF known to interfere with the action of complement C3 factor. NOD.B10-H2 b mice, injected with CVF starting at 10 weeks of age, a time when leukocyte infiltration is expected to begin, failed to develop salivary dysfunction out to 24 weeks of age, a time when reduced salivary flow rates are known to occur in non-treated animals. Concomitant with retention of salivary exocrine function, CVF-treated mice showed reduced levels of leukocytic infiltration, reduction of anti-nuclear autoantibodies and major alterations in the B lymphocyte profiles while maintaining general pathophysiological measures of disease. These data suggest that C3 plays a significant role in development and onset of SjS-like disease, yet additional studies need to be carried out to identify the precise mode of action. [Copyright &y& Elsevier]

Abstract: Introduction: Complement component C3 (C3) can be a target of pharmacological or toxicological agents. In the analysis of this, it is important to examine the involvement of fragments C3b and C3a since C3 function normally requires cleavage into these fragments. The present study describes a simple and efficient method for the preparation of rat complement C3b and C3a by using purified C3 and cobra venom factor (CVF) as a cleaving enzyme. Methods: CVF was purified from lyophilized cobra venom (Naja naja kausia) by two-step chromatography and was activated by incubation with human factors B and D. C3 was cleaved by incubation with activated CVF (CVF,Bb), and C3b and C3a were isolated by anion- and cation-exchange chromatography, respectively. Results: About 200 μg of CVF was purified from100 mg of cobra venom. All the CVF was activated by incubation with factors B and D. The C3b and C3a obtained were pure as analyzed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, and no digestive by-products such as C3f were found. Discussion: The advantage of the present method is that it is possible to prepare relatively large amounts of C3b by simple procedures without digestive by-products. C3a can be prepared from the flow through fraction of the C3b purification. C3b and C3a prepared by the present method would be useful for pharmacological or toxicological experiments involving receptor binding since their binding sites remain intact. [Copyright &y& Elsevier]

The monoglucosylated oligomannose N-linked oligosaccharide (Glc1Man9GlcNAc2) is a retention signal for the calnexin–calreticulin quality control pathway in the endoplasmic reticulum. We report here the presence of such monoglucosylated N-glycans on the human complement serum glycoprotein C3. This finding represents the first report of monoglucosylated glycans on a human serum glycoprotein from non-diseased individuals. The presence of the glucose moiety in 5% of the human C3 glycoprotein suggests that this glycosylation site is sequestered within the protein and is consistent with previous studies identifying a cryptic conglutinin binding site on C3 that becomes exposed upon its conversion to iC3b. [Copyright &y& Elsevier]

Chromium is released during several industrial processes and has accumulated in some estuarine areas. Its effects on mammals have been widely studied, but relatively little information is available on its effects on fish. Gene expression changes are useful biomarkers that can provide information about toxicant exposure and effects, as well as the health of an organism and its ability to adapt to its surroundings. Therefore, we investigated the effects of Cr(VI) on gene expression in the sediment dwelling fish, winter flounder (Pseudopleuronectes americanus). Winter flounder ranging from 300 to 360 g were injected i.p. with Cr(VI) as chromium oxide at 25 μg/kg chromium in 0.15N KCl. Twenty-four hours following injections, winter flounder were euthanized with MS-222 and the livers were excised. Half of the livers were used to make cytosol and the other half were used to isolate mRNA for subtractive hybridization. Subtractive clones obtained were spotted onto nylon filters, which revealed several genes with potentially altered expression due to Cr(VI), including an α class GST, 1-Cys peroxiredoxin (a non-selenium glutathione peroxidase), a P-450 2X subfamily member, two elongation factors (EF-1 gamma and EF-2), and complement component C3. Semi-quantitative RT-PCR was performed and confirmed that Cr(VI) down-regulated complement component C3, an EST, and two potential glutathione peroxidases, GSTA3 and 1-Cys peroxiredoxin. In addition, cytosolic GSH peroxidase activity was reduced, and silver stained SDS-PAGE gels from glutathione-affinity purified cytosol demonstrated that a 27.1 kDa GSH-binding protein was down-regulated greater than 50%. Taken together, Cr(VI) significantly altered the expression of several genes including two potential glutathione peroxidases in winter flounder. [Copyright &y& Elsevier]

The mechanisms that control complement protein synthesis are incompletely understood. Recent evidence suggests that cytokines are involved in the regulation of hepatic synthesis of circulating complement components. Therefore, we compared the effects of human recombinant IL-1α, IL-1β, IL-6, IFN-γ, and TNF-α individually or in combination, on HepG2 secretion of complement component C3, the major opsonic protein of the complement system. HepG2 cells were incubated with each cytokine alone and with various combinations of the cytokines. At 24, 48, 72, and 96 h of incubation, the C3 and albumin secreted by the HepG2 cells were quantified by a sandwich ELISA. IL-1α and IFN-γ significantly enhanced C3 secretion by the cells (P<0.02 vs. control cells). IL-1β when combined with either IL-6 or IFN-γ also increased C3 secretion (P<0.03 vs. control cells). The stimulatory effect on HepG2 cells by the IL-1β/IL-6 combination was synergistic. With the exception of IL-1α, which increased albumin secretion, HepG2 secretion of albumin was not affected by incubation with individual cytokines or the cytokine combinations. Therefore, IL-1α, IFN-γ, and the combination of IL-1β with IL-6 or IFN-γ specifically enhanced C3 secretion by HepG2 cells. The greatest magnitude of C3 secretion was induced by the combination of IL-1β and IL-6. [Copyright &y& Elsevier]

The complement component C3 was isolated from spotted wolffish (Anarhichas minor Olafsen) serum by polyethylene glycol precipitation, anion exchange chromatography and gel filtration. Silver staining in SDS-PAGE and rabbit anti-wolffish C3 antiserum used in Western blotting revealed that spotted wolffish C3 contains two polypeptide chains, Mr65 and 115 kDa, respectively. The high molecular weight α-chain of the C3 incorporated 14C-methylamine suggesting that it contained a reactive thioester group. The deduced amino acid sequence, after screening a liver cDNA expression library, showed that the wolffish C3 contained key amino acids for binding C3 convertase, factor H, I and properdin. Also, high degree of homology to other vertebrate C3 was found in the β–α junction site. Phylogenetic tree analysis indicated that the Japanese flounder and spotted wolffish that belong to order pleuronectiformes and perciformes, respectively, are phylogenetically close species. Immunohistochemical experiments showed that liver hepatocytes and blood contained C3, and in situ hybridisation experiments revealed that liver hepatocytes expressed C3. [Copyright &y& Elsevier]

Anaerobic microorganisms in periodontal pockets produce toxic amounts of hydrogen sulfide. The capacity of polymorphonuclear leukocytes leukocytes to kill a capsulated and a non-capsulated variant of a group B streptococcal strain was studied in presence and absence of sulfide. The killing was equally efficient under aerobic and anaerobic conditions. However, in presence of sulfide the killing of the caqpsulated variant of the strain was significantly inhibited. Since this strain required higher serum concentrations to be killed by the polymorphonuclear leukocytes, it suggested that sulfide interfered with the opsonization of the bacteria. The capacity of sulfide to split the disulfide bonds of complement factor 3 and immunoglobulin G, deposited on the bacterial surface, was evaluated by sodium dedecyl sulfate-polyacrylamide gel electrophoresis. There was no detectable effect of 2 mM sulfide on immunoglobulin G. However, sulfide released from opsonized bacteria the β-chain of C3b C3bi, and the C-terminal part of the α-chain of C3bi. This region of the α-chain of C3bi has been suggested to bind to the complement receptor 3 of polymorphonuclear leukocytes. The β-chain of C3b/C3bi may augment the binding of opsonized bacteria to the complement receptors of polymorphonuclear leukocytes. The formation of sulfide by the microflora of the periodontal pockets may provide conditions for the bacteria to escape important parts of the host immune system. [ABSTRACT FROM AUTHOR]