C3 Activation Research Articles

Aberrant activation of the complement system in renal grafts is mediated by cold storage.

Aberrant complement activation leads to tissue damage during kidney transplantation, and it is recognized as an important target for therapeutic intervention. However, it is not clear whether cold storage (CS) triggers the complement pathway in transplanted kidneys. The goal of the present study was to determine the impact of CS on complement activation in renal transplants. Male Lewis and Fischer rats were used, and donor rat kidneys were exposed to 4 h or 18 h of CS followed by transplantation (CS + transplant). To study CS-induced effects, a group with no CS was included in which the kidney was removed and transplanted back to the same rat [autotransplantation (ATx)]. Complement proteins (C3 and C5b-9) were evaluated with Western blot analysis (reducing and nonreducing conditions) and immunostaining. Western blot analysis of renal extracts or serum indicated that the levels of C3 and C5b-9 increased after CS + transplant compared with ATx. Quite strikingly, intracellular C3 was profoundly elevated within renal tubules after CS + transplant but was absent in sham or ATx groups, which showed only extratubular C3. Similarly, C5b-9 immunofluorescence staining of renal sections showed an increase in C5b-9 deposits in kidneys after CS + transplant. Real-time PCR (SYBR green) showed increased expression of CD11b and CD11c, components of complement receptors 3 and 4, respectively, as well as inflammatory markers such as TNF-α. In addition, recombinant TNF-α significantly increased C3 levels in renal cells. Collectively, these results demonstrate that CS mediates aberrant activation of the complement system in renal grafts following transplantation.NEW & NOTEWORTHY This study highlights cold storage-mediated aberrant activation of complement components in renal allografts following transplantation. Specifically, the results demonstrate, for the first time, that cold storage functions in exacerbation of C5b-9, a terminal cytolytic membrane attack complex, in renal grafts following transplantation. In addition, the results indicated that cold storage induces local C3 biogenesis in renal proximal cells/tubules and that TNF-α promotes C3 biogenesis and activation in renal proximal tubular cells.

Complement C5 is not critical for the formation of sub-RPE deposits in Efemp1 mutant mice

The complement system plays a role in the formation of sub-retinal pigment epithelial (RPE) deposits in early stages of age-related macular degeneration (AMD). But the specific mechanisms that connect complement activation and deposit formation in AMD patients are unknown, which limits the development of efficient therapies to reduce or stop disease progression. We have previously demonstrated that C3 blockage prevents the formation of sub-RPE deposits in a mouse model of EFEMP1-associated macular degeneration. In this study, we have used double mutant Efemp1R345W/R345W:C5-/- mice to investigate the role of C5 in the formation of sub-RPE deposits in vivo and in vitro. The data revealed that the genetic ablation of C5 does not eliminate the formation of sub-RPE deposits. Contrarily, the absence of C5 in RPE cultures promotes complement dysregulation that results in increased activation of C3, which likely contributes to deposit formation even in the absence of EFEMP1-R345W mutant protein. The results also suggest that genetic ablation of C5 alters the extracellular matrix turnover through an effect on matrix metalloproteinases in RPE cell cultures. These results confirm that C3 rather than C5 could be an effective therapeutic target to treat early AMD.

Menthol modified zinc(II) phthalocyanine regioisomers and their photoinduced antimicrobial activity against Staphylococcus aureus

Our study is the first to investigate the antimicrobial activity of the single regioisomers rather than the mixture of isomeric phthalocyanines - regioisomers with symmetry C4, C1 and C2, functionalized with L-menthol in the periphery were obtained. The single Zn(II) phthalocyanine regioisomers were synthesized by remetallation reaction from the metal-free phthalocyanines. The obtained compounds were characterised using NMR, UV–Vis, IR spectroscopy and mass spectrometry. The analysed macrocycles generate singlet oxygen with the quantum yield up to 0.78. Menthol substituted three regioisomers of zinc(II) phthalocyanine were loaded into liposomes and subjected to biological activity study. The investigated compounds present low – C4 and moderate – C1 and C2 activity against Staphylococcus aureus. The highest bacterial growth inhibition (2.48 log) showed regioisomer C2 at the concentration of 10 μM.

Effect of diet enriched with Agaricus bisporus polysaccharides (ABPs) on antioxidant property, innate-adaptive immune response and pro-anti inflammatory genes expression in Ctenopharyngodon idella against Aeromonas hydrophila

The effect of Agaricus bisporus polysaccharides (ABPs) supplemented diet on growth rate, antioxidant capacity, innate-adaptive immune response, proinflammatory and antiinflammatory genes expression in Ctenopharyngodon idella against Aeromonas hydrophila is reported. In both normal and challenged groups fed with 1.0 and 1.5 mg kg−1 ABPs diets resulted in a significant weight gain and feed intake. The survival was 100% in normal fish fed without or with any ABPs diet; the challenged fish fed with 1.0 mg kg−1 ABPs diet had 98.6% survival. The RBC and WBC counts, Hb, and Hct levels were significant in both normal and challenged groups fed with 1.0 and 1.5 mg kg−1 ABPs diets. A significant increase in total protein and albumin level was observed in both groups fed with 1.0 and 1.5 mg kg−1 ABPs diets. Significant increase in GPx, ROS, GR, GSH, PC, and MnSOD activity was observed in HK of both groups fed with 1.0 and 1.5 mg kg−1 ABPs diets; similarly both groups when fed with the same ABPs diets showed significant Lz, C3, and C4 activity. However, both groups fed with 1.0 mg kg−1 ABPs diet showed significant β-defensin, LEAP-2A, IL-6, and NF-κB P65 mRNA expression. Similarly, IFN-γ2, IL-10, and TNFα mRNA expressions were significant in both groups fed with 1.0 mg kg−1 ABPs diet. The results indicate that both normal and challenged C. idella fed with a 1.0 mg kg−1 ABPs diet had better growth, antioxidant status, immune response, and pro-anti-inflammatory gene modulation against A. hydrophila.

Abnormal C5 production and activation contributes to the mortality of COVID-19 patients

Abstract Elucidating the pathogenesis of COVID-19 is vital for treating the disease. Recent clinical studies have suggested that the complement system may play an important role in the course of infection. We investigated the correlation between complement C5, a critical factor in the complement common pathway, and the mortality of COVID-19. Thirty-seven COVID-19 patients were consented for this study. Serial blood samples were collected at different time points from 27 patients who recovered during hospitalization, and 10 patients who did not survive during hospitalization. Circulating native C5 was analyzed by Western Blot to detect α-chain and β-chain of intact C5. The activated C5a levels were detected by an ELISA kit. The levels of C5 β-chain and C5a were significantly higher in the non-survivors than in the survivors. Multivariate regression analyses showed that abnormal levels of C5 β-chain and C5a could independently predict the mortality. Therefore, our results suggest that in COVID-19 patients, the abnormal C5 production and activation may contribute to the systemic inflammation thus affect survival.

Decreased Systemic Complement Activation Product C3a is Associated with a Reduction in Pancreatic β Cell Area in Islets of Female Rat Offspring following Chronic Placental Ischemia‐induced Hypertension

Preeclampsia is a pregnancy specific condition characterized by new-onset maternal hypertension and increased systemic inflammation, including increased complement activation. Our previous studies demonstrated that inhibition of complement activation attenuated placental ischemia-induced hypertension in the Reduced Uteroplacental Perfusion Pressure (RUPP) model of gestational hypertension in rat. Hypertensive pregnancy disorders can also result in increased risk of later-life cardiovascular and metabolic disease for the offspring. In fact, recent studies have demonstrated that gestational hypertension is associated with increased risk for Type 2 diabetes in offspring. Clearly, pancreatic β cell mass, established early in life, can influence whether an individual develops Type 2 diabetes. β cell area has also been linked to complement component C3 and activation product C3a, with increased C3a generation associated with prevention of human islet cell atrophy. Our previous studies demonstrated a significant reduction in β cell area in postnatal day (PD)13 female, but not male, offspring of rats subjected to chronic placental ischemia-induced hypertension in the RUPP model. Thus, we hypothesized that decreased C3a is associated with reductions in β cell area in the female offspring following placental insufficiency. The RUPP model was used to induce placental insufficiency by placing silver clips on the abdominal aorta and uterine arteries on gestation day 14 of a 21 day gestation. Serum was collected via the abdominal aorta from PD13 pups born from RUPP and Sham pregnancies. Pancreatic islets of PD13 male and female offspring of RUPP and Sham dams were isolated by collagenase perfusion and handpicking. We previously demonstrated that the transcript message for membrane-bound complement regulator CD55 (negative regulator of C3 activation) was significantly increased in islets isolated from female, but not male, RUPP vs Sham offspring, with no change in acinar tissue. In addition, no significant change was observed in message for the parent molecule C3. The relative amount of circulating C3a in serum of PD13 offspring was determined by Western blot and expressed as C3a units/ul based on signal intensity of 1 ul of a standard pool of adult rat serum activated by yeast. Circulating C3a in female RUPP offspring (1.35 ± 0.37 units/ul) was significantly lower (p<0.05) than C3a in female Sham offspring (2.54 ± 0.44 units/ul). No difference was noted comparing C3a in serum of male offspring, RUPP vs Sham, where no change in β cell area has been observed. Thus, these data suggest an increase in CD55 may limit complement activation and C3a production in female offspring. This dysregulation of complement activation may contribute to reduction of β cell area and long-term susceptibility for Type 2 diabetes. Future studies are needed to determine if local or circulating C3a is instrumental in controlling pancreatic β cell area in offspring of hypertensive pregnancies.

The Multifunctional Sactipeptide Ruminococcin C1 Displays Potent Antibacterial Activity In Vivo as Well as Other Beneficial Properties for Human Health.

The world is on the verge of a major antibiotic crisis as the emergence of resistant bacteria is increasing, and very few novel molecules have been discovered since the 1960s. In this context, scientists have been exploring alternatives to conventional antibiotics, such as ribosomally synthesized and post-translationally modified peptides (RiPPs). Interestingly, the highly potent in vitro antibacterial activity and safety of ruminococcin C1, a recently discovered RiPP belonging to the sactipeptide subclass, has been demonstrated. The present results show that ruminococcin C1 is efficient at curing infection and at protecting challenged mice from Clostridium perfringens with a lower dose than the conventional antibiotic vancomycin. Moreover, antimicrobial peptide (AMP) is also effective against this pathogen in the complex microbial community of the gut environment, with a selective impact on a few bacterial genera, while maintaining a global homeostasis of the microbiome. In addition, ruminococcin C1 exhibits other biological activities that could be beneficial for human health, as well as other fields of applications. Overall, this study, by using an in vivo infection approach, confirms the antimicrobial clinical potential and highlights the multiple functional properties of ruminococcin C1, thus extending its therapeutic interest.

Complement and kidney disease, new insights.

In this review, we discuss recent studies showing the importance of the complement pathway in kidney disease. Recent findings in C3 glomerulopathy (C3G) include: acute postinfectious glomerulonephritis is characterised by the presence of antifactor B antibodies; human leukocyte antigen type, but not rare complement gene variation, is associated with primary immunoglobulin-associated membranoproliferative GN and C3G. Immunohistochemistry in C3G shows that factor H related protein 5 (FHR5) is the most prevalent complement protein and correlates with kidney function. A multicentre study supported the use of mycophenolate mofetil (MMF) in C3G even after a propensity matching analysis. In immunoglobulin A nephropathy (IgAN) several studies have emphasised the importance of complement. Imbalances of circulating FH and FHR1 and FHR5, which interfere with the regulatory functions of FH, associate with IgAN. Immunohistochemistry has shown associations between glomerular FHR5 deposition and C3 activation; glomerular FHR5 associated with clinical markers of IgAN severity. Data also suggest the lectin complement pathway contributes to IgAN severity. We also discuss complement activation in thrombotic microangiopathy and other kidney diseases. Complement activity can be detected in a wide range of kidney diseases and this provides pathogenic insight and potential for therapy with the ongoing development of several drugs directed at complement activation.

Complement Profiles in Patients with Amyotrophic Lateral Sclerosis: A Prospective Observational Cohort Study.

BackgroundThe complement system has been suggested to be involved in the pathophysiology of amyotrophic lateral sclerosis (ALS), a progressive motor neuron disease. In the present study, we compared levels of selected complement markers to clinical outcome in ALS patients.MethodsThis observational, explorative cohort study included 92 ALS patients, 61 neurological controls (NCs) admitted for suspected aneurysmal subarachnoid haemorrhage, and 96 neurologically healthy controls (NHCs). Peripheral blood and cerebrospinal fluid (CSF) were obtained for the measurement of ficolin-1, −2, and −3; collectin-11, MBL, MASP-3, MAP-1, C4, C3, PTX-3, and complement activation products C4c, C3bc, and sC5b-9. We recorded clinical outcomes of ALS patients for 24 to 48 months after inclusion in order to analyse the effects of the complement markers on survival time.ResultsCompared with both control groups, ALS patients exhibited increased collectin-11, C4 and sC5b-9 in plasma, as well as increased ficolin-3 in CSF. Ficolin-2 was significantly decreased in plasma of the ALS patients compared with NHCs, but not with NCs. The concentration of collectin-11, C3 and C3bc correlated negatively with the revised ALS functional rating scale (ALSFRS-R). No association was found between levels of complement markers and survival as estimated by hazard ratios.ConclusionALS patients exhibit aberrant expression of selected mediators of the lectin complement pathway as well as increased activation of the terminal complement pathway, corroborating the notion that the complement system might be involved in the pathophysiology of ALS.

The M domain in UNC-13 regulates the probability of neurotransmitter release

SUMMARYSynapses exhibit multiple forms of short-term plasticities, which have been attributed to the heterogeneity of neurotransmitter release probability. However, the molecular mechanisms that underlie the differential release states remain to be fully elucidated. The Unc-13 proteins appear to have key roles in synaptic function through multiple regulatory domains. Here, we report that deleting the M domain in Caenorhabditis elegans UNC-13MR leads to a significant increase in release probability, revealing an inhibitory function of this domain. The inhibitory effect of this domain is eliminated when the C1 and C2B domains are absent or activated, suggesting that the M domain inhibits release probability by suppressing the activity of C1 and C2B domains. When fused directly to the MUNC2C fragment of UNC-13, the M domain greatly enhances release probability. Thus, our findings reveal a mechanism by which the UNC-13 M domain regulates synaptic transmission and provides molecular insights into the regulation of release probability.

A novel injury site-natural antibody targeted complement inhibitor protects against lung transplant injury.

Complement is known to play a role in ischemia and reperfusion injury (IRI). A general paradigm is that complement is activated by self-reactive natural IgM antibodies (nAbs), after they engage postischemic neoepitopes. However, a role for nAbs in lung transplantation (LTx) has not been explored. Using mouse models of LTx, we investigated the role of two postischemic neoepitopes, modified annexin IV (B4) and a subset of phospholipids (C2), in LTx. Antibody deficient Rag1-/- recipient mice were protected from LTx IRI. Reconstitution with either B4 or C2nAb restored IRI, with C2 significantly more effective than B4 nAb. Based on these information, we developed/characterized a novel complement inhibitor composed of single-chain antibody (scFv) derived from the C2 nAb linked to Crry (C2scFv-Crry), a murine inhibitor of C3 activation. Using an allogeneic LTx, in which recipients contain a full nAb repertoire, C2scFv-Crry targeted to the LTx, inhibited IRI, and delayed acute rejection. Finally, we demonstrate the expression of the C2 neoepitope in human donor lungs, highlighting the translational potential of this approach.

Inhibition of the Complement Alternative Pathway by Chemically Modified DNA Aptamers That Bind with Picomolar Affinity to Factor B.

The complement system is a conserved component of innate immunity that fulfills diverse roles in defense and homeostasis. Inappropriate activation of complement contributes to many inflammatory diseases, however, which has led to a renewed emphasis on development of therapeutic complement inhibitors. Activation of complement component C3 is required for amplification of complement and is achieved through two multisubunit proteases called C3 convertases. Of these, the alternative pathway (AP) C3 convertase is responsible for a majority of the C3 activation products in vivo, which renders it an attractive target for inhibitor discovery. In this study, we report the identification and characterization of two related slow off-rate modified DNA aptamers (SOMAmer) reagents that inhibit formation of the AP C3 convertase by binding to the proprotease, factor B (FB). These aptamers, known as SL1102 (31 bases) and SL1103 (29 bases), contain uniform substitutions of 5-(N-2-naphthylethylcarboxyamide)-2'-deoxyuridine for deoxythymidine. SL1102 and SL1103 bind FB with K d values of 49 and 88 pM, respectively, and inhibit activation of C3 and lysis of rabbit erythrocytes under AP-specific conditions. Cocrystal structures of SL1102 (3.4 Å) and SL1103 (3.1 Å) bound to human FB revealed that SL1102 and SL1103 recognize a site at the juncture of the CCP1, CCP3, and vWF domains of FB. Consistent with these structures and previously published information, these aptamers inhibited FB binding to C3b and blocked formation of the AP C3 convertase. Together, these results demonstrate potent AP inhibition by modified DNA aptamers and expand the pipeline of FB-binding molecules with favorable pharmacologic properties.

Hepatitis B Virus Infection Among Leprosy Patients: A Case for Polymorphisms Compromising Activation of the Lectin Pathway and Complement Receptors.

Thousands of leprosy patients not only suffer from physical deformities, but also either have or have had hepatitis B virus (HBV) coinfection. Polymorphisms of the complement system modulate susceptibility to leprosy, but genetic susceptibility to past or present HBV infection is unknown. We used sequencing and multiplex sequence-specific PCR to genotype 72 polymorphisms of seven genes (MBL2, FCN1, FCN2, FCN3, MASP1, MASP2, C3) encoding components of the lectin pathway, and two genes encoding complement receptors (CR1, VSIG4) in 190 patients, of which 74 were positive for HBsAg and/or anti-HBc (HBV+, 93.2% with a resolved infection) and 116 lepromatous patients, and 408 HBV-blood donors. In addition, we tested for levels of proteins of the lectin pathway. We found no difference between serum concentrations of mannan-binding lectin (MBL), MBL-associated serine proteins (MASP-1, MASP-2, MASP-3, MAp44), ficolin-3 (FCN-3), soluble complement receptor 1 (sCR1) and MBL mediated C4 activation, measured by ELISA or TRIFMA in up to 167 HBV+ and HBV− patients. Haplotypes lowering protein levels or encoding dysfunctional proteins increased susceptibility to HBV infection: MBL2*LYQC (OR = 3.4, p = 0.02), MASP1*AC_CC (OR = 4.0, p = 0.015) and MASP2*1C2-l (OR = 5.4, p = 0.03). Conversely, FCN1*3C2 haplotype, associated with higher gene expression, was protective (OR = 0.56, P = 0.033). Other haplotypes associated with HBV susceptibility were: MASP2*2B1-i (OR = 19.25, P = 0.003), CR1*3A (OR = 2.65, P = 0.011) and VSIG4*TGGRCG (OR = 12.55, P = 0.014). Some polymorphisms in ficolin genes associated with lower protein levels increased susceptibility to leprosy/HBV infection: FCN*1 (OR = 1.66, P = 0.029), FCN2*GGGCAC (OR = 6.73, P = 0.008), and FCN3*del_del_C (OR = 12.54, P = 0.037), and to lepromatous disease/HBV infection: FCN2*TA (OR = 2.5, P = 0.009), whereas FCN2*MAG was associated with increased FCN-2 expression and resistance against coinfection (OR = 0.29, P = 0.026). These associations were independent of demographic factors and did not increase susceptibility to leprosy per se, except MASP2*1C2-l. Associations for FCN2, FCN3, MASP1, MASP2, and VSIG4 variants were also independent of each other. In conclusion, polymorphisms compromising activation of the lectin pathway of complement increase susceptibility to HBV infection, with ficolin polymorphisms playing a major role in modulating the susceptibility among leprosy patients.

Complement inhibition at the level of C3 or C5: mechanistic reasons for ongoing terminal pathway activity

AbstractBlocking the terminal complement pathway with the C5 inhibitor eculizumab has revolutionized the clinical management of several complement-mediated diseases and has boosted the clinical development of new inhibitors. Data on the C3 inhibitor Compstatin and the C5 inhibitors eculizumab and Coversin reported here demonstrate that C3/C5 convertases function differently from prevailing concepts. Stoichiometric C3 inhibition failed to inhibit C5 activation and lytic activity during strong classical pathway activation, demonstrating a “C3 bypass” activation of C5. We show that, instead of C3b, surface-deposited C4b alone can also recruit and prime C5 for consecutive proteolytic activation. Surface-bound C3b and C4b possess similar affinities for C5. By demonstrating that the fluid phase convertase C3bBb is sufficient to cleave C5 as long as C5 is bound on C3b/C4b-decorated surfaces, we show that surface fixation is necessary only for the C3b/C4b opsonins that prime C5 but not for the catalytic convertase unit C3bBb. Of note, at very high C3b densities, we observed membrane attack complex formation in absence of C5-activating enzymes. This is explained by a conformational activation in which C5 adopts a C5b-like conformation when bound to densely C3b-opsonized surfaces. Stoichiometric C5 inhibitors failed to prevent conformational C5 activation, which explains the clinical phenomenon of residual C5 activity documented for different inhibitors of C5. The new insights into the mechanism of C3/C5 convertases provided here have important implications for the development and therapeutic use of complement inhibitors as well as the interpretation of former clinical and preclinical data.

Artesunate: A natural product-based immunomodulator involved in human complement

Complement is an important innate immune defence machinery. Once dysregulated, it is often linked to pathogenesis of diverse autoimmune diseases. Artesunate (ART) is a well-known anti-malarial compound. Recently, ART has been highlighted by its potential therapeutic effects on certain complement-related autoimmune diseases. However, the underlying mechanisms are hitherto unknown. In the present study, we found that ART mediated complement interception as validated by analysis of complement haemolytic assay. In cell-based setup using dying Jurkat cells, ART-mediated complement interception was also confirmed. Further, we newly established an ELISA system selectively allowing complement activation via the classical pathway, the lectin pathway and the alternative pathway, respectively. ELISA analysis revealed that ART dose-dependently inhibited C4 activation, C3 activation and terminal complement complex assembly via the effector pathways. ART was found to blockade C1q, C3 and C5 with a lesser extent to properdin. The interaction of ART with C1q was determined to be mediated via C1q globular head region. FACS analysis using ART-conjugated mesoporous silica particles revealed that ART specifically bound the key therapeutic targets of C1q, C3 and C5 on microparticles. In conclusion, we for the first time report the anti-complement bioactivities of ART and suggest a potential therapeutic benefit of ART in the complement-related human diseases.

Biomarkers of disease activity in patients with chronic spontaneous urticaria.

IntroductionPrevious studies have examined biomarkers of coagulation, inflammation and immunity in chronic spontaneous urticaria (CSU), but no recommended biomarkers for disease activity have been established yet.AimTo find the relationship between certain laboratory parameters and disease activity in patients with CSU.Material and methodsSerum concentrations of D-dimer, C-reactive protein (CRP), C3, C4, and prothrombin time (PT), activated partial thromboplastin time (aPTT) values were measured in 44 CSU patients and compared with 33 healthy controls. Correlation between biomarkers and urticaria activity score during 7 consecutive days (UAS7) was calculated.ResultsOur study included 44 CSU patients (38 females and 6 males), mean age of 50.4 years and the average disease duration of 3.1 years. Based on UAS7, 23 (52.3%) CSU patients had mild urticaria, 8 (18.2%) well-controlled, 7 (15.9%) moderate and 6 (13.6%) severe urticaria. Fourteen (31.8%) patients had elevated CRP, 21 (47.7%) had elevated D-dimer and 14 (13.6%) CSU patients had elevated C4 levels. Patients with CSU had statistically significant elevated D-dimer, CRP and PT as compared with controls (p = 0.007, p = 0.005 and p = 0.029, respectively). There was no correlation between PT, aPTT, D-dimer, CRP, C3 and disease activity. Statistically significant differences in C4 levels between patients with severe and well-controlled, mild, moderate urticaria were determined (p = 0.003).ConclusionsCRP, D-dimer, and PT may be considered as biomarkers for distinguishing patients with CSU from controls. The C4 levels correlate with disease activity and may be useful as a potential biomarker of disease activity.

Kan ve Oral Kavite Örneklerinden Soyutlanan Candida albicans Suşlarının Fosfolipaz Aktivitelerinin Araştırılması

Objective: The aim of the present study was to investigate and compare the phospholipase B1, B2, C1 and D1 activities in C.albicans strains isolated from blood cultures and oral cavity specimens. Method: Phospholipase activity of the strains was examined by plate method and reverse transcriptase polymerase chain reaction (RT-PZT). Results: Twenty-six (86.7%) strains isolated from blood and 24 (80.0%) from oral cavities revealed phospholipase activity by plate method. No statistically significant difference (χ2 =0.48; p=0.49) was observed between the groups. However statistical difference was determined between the mean Pz values (0.6138±0.9823; 0.6988±0.9910) (p=0.007). PLB1 expression was detected in all (100%), PLB2 in 29 (96.7%) and 30 (100.0%), PLC1 in 27 (90%) and 22 (73.3%), PLD1 in 27 (90.0%) and 21 (70.0%) of blood and oral strains, respectively. While no significant difference was detected between PLB1 expression values of the groups (t=-0.307; p=0.760), PLB2 and PLC1 expressions were found to be significantly higher in blood (p=0.043) and oral cavity isolates (p&lt;0.001), respectively. No difference was observed between PLD1 expressions (p=0.732). PLB1, PLB2, PLC1 and PLD1 expressions were 100%, 81.7%, 71.6% and 76.7% in agreement with the plate method. The agreement was 83.3% when all the phospholipase genes were considered. No correlation was detected between the Pz values and phospholipase expressions (p=0.602; p=0.555; p=0.241; p=0,096, respectively). Conclusion: The high rates of phospholipase activity of the C.albicans isolates in our study, support the important roles of these enzymes in virulence. Our results may indicate that phospholipase enzymes encoded by PLB2 and PLC1 genes play more important roles for invasive and oral cavity infections, respectively; however large scale studies are needed on this issue.

An electrochemical method for detecting the biomarker 4-HPA by allosteric activation of Acinetobacterbaumannii reductase C1 subunit

The C1 (reductase) subunit of 4-hydroxy-phenylacetate (4-HPA) 3-hydroxylase (HPAH) from the soil-based bacterium Acinetobacterbaumannii catalyzes NADH oxidation by molecular oxygen, with hydrogen peroxide as a by-product. 4-HPA is a potent allosteric modulator of C1, but also a known urinary biomarker for intestinal bacterial imbalance and for some cancers and brain defects. We thus envisioned that C1 could be used to facilitate 4-HPA detection. The proposed test protocol is simple and in situ and involves addition of NADH to C1 in solution, with or without 4-HPA, and direct acquisition of the H2O2 current with an immersed Prussian Blue–coated screen-printed electrode (PB-SPE) assembly. We confirmed that cathodic H2O2 amperometry at PB-SPEs is a reliable electrochemical assay for intrinsic and allosterically modulated redox enzyme activity. We further validated this approach for quantitative NADH electroanalysis and used it to evaluate the activation of NADH oxidation of C1 by 4-HPA and four other phenols. Using 4-HPA, the most potent effector, allosteric activation of C1 was related to effector concentration by a simple saturation function. The use of C1 for cathodic biosensor analysis of 4-HPA is the basis of the development of a simple and affordable clinical routine for assaying 4-HPA in the urine of patients with a related disease risk. Extension of this principle to work with other allosteric redox enzymes and their effectors is feasible.

Antioxidant and UV-radiation absorption activity of aaptamine derivatives - potential application for natural organic sunscreens.

Antioxidant and UV absorption activities of three aaptamine derivatives including piperidine[3,2-b]demethyl(oxy)aaptamine (C1), 9-amino-2-ethoxy-8-methoxy-3H-benzo[de][1,6]naphthyridine-3-one (C2), and 2-(sec-butyl)-7,8-dimethoxybenzo[de]imidazo[4,5,1-ij][1,6]-naphthyridin-10(9H)-one (C3) were theoretically studied by density functional theory (DFT). Direct antioxidant activities of C1–C3 were firstly evaluated via their intrinsic thermochemical properties and the radical scavenging activity of the potential antioxidants with the HOO˙/HO˙ radicals via four mechanisms, including: hydrogen atom transfer (HAT), single electron transfer (SET), proton loss (PL) and radical adduct formation (RAF). Kinetic calculation reveals that HOO˙ scavenging in water occurs via HAT mechanism with C1 (kapp, 7.13 × 106 M−1 s−1) while RAF is more dominant with C2 (kapp, 1.40 × 105 M−1 s−1) and C3 (kapp, 2.90 × 105 M−1 s−1). Antioxidant activity of aaptamine derivatives can be classified as C1 > C3 > C2. Indirect antioxidant properties based on Cu(i) and Cu(ii) ions chelating activity were also investigated in aqueous phase. All three studied compounds show spontaneous and favorable Cu(i) ion chelating activity with ΔG0 being −15.4, −13.7, and −15.7 kcal mol−1, whereas ΔG0 for Cu(ii) chelation are −10.4, −10.8, and −2.2 kcal mol−1 for C1, C2 and C3, respectively. In addition, all compounds show UVA and UVB absorption; in which the excitations are determined mostly as π–π* transition. Overall, the results suggest the potential applications of the aaptamines in pharmaceutics and cosmetics, i.e. as a sunscreen and antioxidant ingredient.

C3a and Its Receptor C3aR Are Detectable in Normal Human Epidermal Keratinocytes and Are Differentially Regulated via TLR3 and LL37

To study the molecular interplay between TLRs and complement representing ancient danger-sensing mechanisms, we examined the regulation of the C3a/anaphylatoxin C3a receptor (C3aR) axis in normal human epidermal keratinocytes (NHEKs) by treatment with different TLR ligands. Protein staining followed by flow cytometry revealed highly constitutive intracellular expression levels of the C3aR in NHEKs. Stimulation with Poly I:C up-regulated C3aR mRNA and intra- and extracellular expression in NHEKs which showed functional relevance by up-regulating CXCL10 and down-regulating C3 expression in response to C3a. mRNA and protein levels of C3 and protease cathepsin L (CTSL) that can cleave C3 were up-regulated by the TLR3 ligand Poly I:C. Enhanced intracellular expression levels of the biologically active C3 fragment (C3a), in response to TLR3 stimulation were also detectable in NHEKs. Cathelicidin antimicrobial peptide LL-37 potentiated Poly I:C-induced C3aR, C3, and CTSL up-regulation. In conclusion, we point to a role of TLR3 to promote up-regulation of C3aR, C3, and CTSL expression levels and generation of C3a. Our data provide evidence that local generation and activation of complement components as described for T cells or myeloid cells represent a scenario which may take place in a similar way in NHEKs.