Complement systems C4, C3 and CH50 not subject to a circadian rhythm.

BACKGROUND: The circadian fluctuations in the blood levels of selected components of the complement system are ill-defined. Some authors found nadir serum levels of C4 and C3 components, together with C3a at nighttime, while others reported insomnia when pro-inflammatory components exhibited increased serum levels. In this study, we quantitatively estimate the morning and evening daytime serum levels of CH50, C4, C3, put into context with C-reactive protein (CRP), cortisol, parathyroid hormone (PTH) and 25(OH)vitamin D at 07:00 A.M. and at 07:00 P.M. METHODS: Seven healthy adult women and 11 men who were voluntary participants agreed to a fasting venipuncture in the morning after having normally eaten through the day and in the evening. The C4 and C3 serum levels were measured on a Cobas (Roche Diagnostics, Switzerland) modular analyzer, CH50 was estimated using the COMPL300 enzyme-linked immunosorbent assay (ELISA) of Wieslab (Malmö, Sweden). CRP, 25(OH)vitamin D, PTH and cortisol concentrations were assessed with electro-chemiluminescence immunoassay (ECLIA) on the Roche Cobas 6000 platform; IgG was measured using nephelometry (Siemens, Germany). RESULTS: With the exception of higher PTH levels in the evening [3.12-5.46, 95% confidence interval (CI)] compared to the morning (2.93-4.65, 95% CI), the mean and median values of C4, C3, CH50 as well as CRP, PTH and 25(OH)vitamin D fell within the established reference intervals. Cortisol levels were measured as an internal positive control for diurnal fluctuations (morning: 294-522 nmol/L, 95% CI; evening: 106-136 nmol/L, 95% CI). CONCLUSIONS: The concentrations of the assessed complement components C4 and C3 as well as CH50 surrogate assay did not yield significantly different values between early morning and evening. This does not exclude their participation in the circadian metabolome; this pilot study with healthy participants suggests that patients with an autoimmune disease in remission can give their blood samples independently during daytime with or without fasting.

Anti-red blood cell antibodies, free light chains, and antiphospholipid antibodies in intravenous immunoglobulin preparations.

BACKGROUND: Anti-red blood cell antibodies, free light chains (FLC) and prothrombotic proteins (PTP) may co-elute with intact immunoglobulin (IgG), and may be the cause of adverse reactions to intravenous immunoglobulin preparations (IVIG). OBJECTIVES: To investigate the presence of residual amounts of these components in IVIG and their effects on ABO blood group agglutination. METHODS: Iso-agglutinin anti-A and anti-B activity was determined with a direct hemagglutination assay of red blood cell (RBC) suspensions from 1% of 46 blood donors together with the serial dilutions of five IVIG (IV1, IV2, IV3, IV4, IV5). Anti-A1 monoclonal antibody was used to confirm reactivity with the A1-reference RBC. The selected IVIG were diluted to a final concentration of 25 mg/ml in 0.15 M NaCI and 0.01 M phosphate-buffered saline, pH 7.4, with or without a further twofold dilution in a low ionic strength solution. RESULTS: A variation up to fivefold in the titer strength of anti-A/B activity was observed between the IVIG preparations. A2-type RBC required higher IVIG inputs when tested in 0.15 M NaCl. The differences in FLC kappa and lambda concentrations were as high as > 400 mg/L among the various IVIG. Only IV1 had a significantly high level of antiphospholipid IgG antibodies (18 U/ml). We demonstrated the presence of anti-RBC antibodies, FLC and PTP in IVIG preparations. CONCLUSIONS: Our findings provide clear evidence that IVIG may harbor pathophysiological substrates with a potential risk for adverse effects such as iatrogenic hemolysis, FLC-associated disorders, and thromboembolism.

Dextran sulfate modulates MAP kinase signaling and reduces endothelial injury in a rat aortic clamping model.

OBJECTIVE: Mitogen-activated protein kinases (MAPKs), including JNK, p38, and ERK1/2, noticeably influence ischemia/reperfusion injury (IRI). The complement inhibitor dextran sulfate (DXS) associates with damaged endothelium denudated of its heparan sulfate proteoglycan (HSPG) layer. Other glycosaminoglycan analogs are known to influence MAPK signaling. Hypothetically therefore, targeted intravascular cytoprotection by DXS may function in part through influencing MAPK activation to reduce IRI-induced damage of the vasculature. METHODS: IRI of the infrarenal aorta of male Wistar rats was induced by 90 minutes clamping followed by 120 minutes reperfusion. DXS (5 mg/mL) or physiologic saline (NaCl controls) was infused locally into the ischemic aortic segment immediately prior to reperfusion. Ninety minutes ischemia-only and heparinase infusion (maximal damage) experiments, as well as native rat aorta, served as controls. Aortas were excised following termination of the experiments for further analysis. RESULTS: DXS significantly inhibited IRI-induced JNK and ERK1/2 activation (P = .043; P =.005) without influencing the p38 pathway (P =.110). Reduced aortic injury, with significant inhibition of apoptosis (P = .032 for DXS vs NaCl), correlated with decreased nuclear factor kappaB translocation within the aortic wall. DXS treatment clearly reduced C1q, C4b/c, C3b/c, and C9 complement deposition, whilst preserving endothelial cell integrity and reducing reperfusion-induced HSPG shedding. Protection was associated with binding of fluorescein labeled DXS to ischemically damaged tissue. CONCLUSIONS: Local application of DXS into ischemic vasculature immediately prior to reperfusion reduces complement deposition and preserves endothelial integrity, partially through modulating activation of MAPKs and may offer a new approach to tackle IRI in vascular surgical procedures. CLINICAL RELEVANCE: The purpose of the present study was to determine the role of dextran sulfate (DXS), a glycosaminoglycan analog and complement inhibitor, in modulating intracellular MAPK signaling pathways, reducing complement activation and ultimately attenuating ischemia/reperfusion injury (IRI) in a rat aortic-clamping model, in part a surrogate model to study the microvasculature. The study shows a role for DXS in ameliorating endothelial injury by reducing IRI-mediated damage and intravascular, local inflammation in the affected aortic segment. DXS may be envisaged as an endothelial protectant in vascular injury, such as occurs during vascular surgical procedures.

Eotaxin/CCL11 expression by infiltrating macrophages in rat heart transplants during ongoing acute rejection.

Eotaxin/CCL11 chemokine is expressed in different organs, including the heart, but its precise cellular origin in the heart is unknown. Eotaxin is associated with Th2-like responses and exerts its chemotactic effect through the chemokine receptor-3 (CCR3), which is also expressed on mast cells (MC). The aim of our study was to find the cellular origin of eotaxin in the heart, and to assess whether expression is changing during ongoing acute heart transplant rejection, indicating a correlation with mast cell infiltration which we observed in a previous study. In a model of ongoing acute heart transplant rejection in the rat, we found eotaxin mRNA expression within infiltrating macrophages, but not in mast cells, by in situ-hybridization. A five-fold increase in eotaxin protein in rat heart transplants during ongoing acute rejection was measured on day 28 after transplantation, compared to native and isogeneic control hearts. Eotaxin concentrations in donor hearts on day 28 after transplantation were significantly higher compared to recipient hearts, corroborating an origin of eotaxin from cells within the heart, and not from the blood. The quantitative comparison of eotaxin mRNA expression between native hearts, isografts, and allografts, respectively, revealed no statistically significant difference after transplantation, probably due to an overall increase in the housekeeping gene's 18S rRNA during rejection. Quantitative RT-PCR showed an increase in mRNA expression of CCR3, the receptor for eotaxin, during ongoing acute rejection of rat heart allografts. Although a correlation between increasing eotaxin expression by macrophages and mast cell infiltration is suggestive, functional studies will elucidate the role of eotaxin in the process of ongoing acute heart transplant rejection.

Restoration of hepatic mast cells and expression of a different mast cell protease phenotype in regenerating rat liver after 70%-hepatectomy.

Mast cells (MC) can undergo significant changes in number and phenotype; these alterations result in the differential expression of growth factors and cytokines. Kit ligand (KL; stem cell factor) is produced by mesenchymal cells, and in the liver by biliary epithelial cells. Recent studies suggest that KL, and its receptor c-kit, may be involved in liver regeneration after loss of liver mass. However, KL is also the major growth, differentiating, chemotactic, and activating factor for MC. The aim of our study was to elucidate the dynamics and phenotype of hepatic MC and KL/c-kit expression during liver regeneration after partial (70%) hepatectomy in the rat. Regenerating livers were harvested after 1, 3, 7, and 14 days, respectively (n = 6 each day). MC were stained for naphthol-AS/D-chloroacetate esterase and counted as MC per bile ductule. MC phenotype was assessed by rat MC protease (RMCP)-1 and -2 immunofluorescence staining, in order to distinguish RMCP-1 positive connective tissue MC (CTMC) from RMCP-2 positive mucosa MC (MMC). mRNA expression of RMCP, c-kit, and the differentially spliced variants of KL was quantified by RT-PCR. MC counts per bile ductule decreased in regenerating rat liver tissue at day 3, compared with native livers, and became normal thereafter. Hepatic MC were predominantly of a CTMC phenotype expressing RMCP-1, as previously published; after hepatectomy, between 76 and 99% of all MC double-expressed RMCP-1 and -2, compatible with an MMC phenotype. The ratio of the two alternatively spliced mRNAs for KL (KL-1 : KL-2), and c-kit mRNA expression did not differ significantly between regenerating livers and the livers of sham operated animals. These results suggest that hepatic mast cells are restored during liver regeneration after partial hepatectomy in the rat. Restored MC express an MMC phenotype, suggesting migration from outside into the regenerating liver. Alternative splicing of KL is affected by the surgical procedure in general, and, together with its receptor c-kit, doesn't seem to be involved in liver regeneration after partial hepatectomy in the rat. Further functional studies, and studies in regenerating human livers might offer the possibility of elucidating the role of the hepatic mast cell, and its different protease phenotypes during liver regeneration after surgical loss of liver mass.

Multimeric tyrosine sulfate acts as an endothelial cell protectant and prevents complement activation in xenotransplantation models.

BACKGROUND: Activation of endothelial cells (EC) in xenotransplantation is mostly induced through binding of antibodies (Ab) and activation of the complement system. Activated EC lose their heparan sulfate proteoglycan (HSPG) layer and exhibit a procoagulant and pro-inflammatory cell surface. We have recently shown that the semi-synthetic proteoglycan analog dextran sulfate (DXS, MW 5000) blocks activation of the complement cascade and acts as an EC-protectant both in vitro and in vivo. However, DXS is a strong anticoagulant and systemic use of this substance in a clinical setting might therefore be compromised. It was the aim of this study to investigate a novel, fully synthetic EC-protectant with reduced inhibition of the coagulation system. METHOD: By screening with standard complement (CH50) and coagulation assays (activated partial thromboplastin time, aPTT), a conjugate of tyrosine sulfate to a polymer-backbone (sTyr-PAA) was identified as a candidate EC-protectant. The pathway-specificity of complement inhibition by sTyr-PAA was tested in hemolytic assays. To further characterize the substance, the effects of sTyr-PAA and DXS on complement deposition on pig cells were compared by flow cytometry and cytotoxicity assays. Using fluorescein-labeled sTyr-PAA (sTyr-PAA-Fluo), the binding of sTyr-PAA to cell surfaces was also investigated. RESULTS: Of all tested compounds, sTyr-PAA was the most effective substance in inhibiting all three pathways of complement activation. Its capacity to inhibit the coagulation cascade was significantly reduced as compared with DXS. sTyr-PAA also dose-dependently inhibited deposition of human complement on pig cells and this inhibition correlated with the binding of sTyr-PAA to the cells. Moreover, we were able to demonstrate that sTyr-PAA binds preferentially and dose-dependently to damaged EC. CONCLUSIONS: We could show that sTyr-PAA acts as an EC-protectant by binding to the cells and protecting them from complement-mediated damage. It has less effect on the coagulation system than DXS and may therefore have potential for in vivo application.

Endothelial cell protection by dextran sulfate: a novel strategy to prevent acute vascular rejection in xenotransplantation.

We showed recently that low molecular weight dextran sulfate (DXS) acts as an endothelial cell (EC) protectant and prevents human complement- and NK cell-mediated cytotoxicity towards porcine cells in vitro. We therefore hypothesized that DXS, combined with cyclosporine A (CyA), could prevent acute vascular rejection (AVR) in the hamster-to-rat cardiac xenotransplantation model. Untreated, CyA-only, and DXS-only treated rats rejected their grafts within 4-5 days. Of the hearts grafted into rats receiving DXS in combination with CyA, 28% survived more than 30 days. Deposition of anti-hamster antibodies and complement was detected in long-term surviving grafts. Combined with the expression of hemoxygenase 1 (HO-1) on graft EC, these results indicate that accommodation had occurred. Complement activity was normal in rat sera after DXS injection, and while systemic inhibition of the coagulation cascade was observed 1 h after DXS injection, it was absent after 24 h. Moreover, using a fluorescein-labeled DXS (DXS-Fluo) injected 1 day after surgery, we observed a specific binding of DXS-Fluo to the xenograft endothelium. In conclusion, we show here that DXS + CyA induces long-term xenograft survival and we provide evidence that DXS might act as a local EC protectant also in vivo.

Dextran sulfate acts as an endothelial cell protectant and inhibits human complement and natural killer cell-mediated cytotoxicity against porcine cells.

BACKGROUND: The innate immune system, including complement and natural killer (NK) cells, plays a critical role in activation and damage of endothelial cells (ECs) during xenograft rejection. The semisynthetic proteoglycan analog dextran sulfate (DXS, molecular weight 5,000) is known to inhibit the complement and coagulation cascades. We hypothesized that DXS may act as an "EC-protectant" preventing complement and NK lysis by functionally replacing heparan sulfate proteoglycans that are shed from the EC surface on activation of the endothelium. METHODS: Binding of DXS to ECs, deposition of human complement, cytotoxicity, and heparan sulfate expression after exposure to normal human serum were analyzed by flow cytometry. The efficacy of DXS to protect ECs from xenogeneic NK cell-mediated cytotoxicity was tested in standard 51Cr-release assays. RESULTS: DXS dose-dependently inhibited all three pathways of complement activation. Binding of DXS to porcine cells increased on treatment with human serum or heparinase I and correlated positively with the inhibition of human complement deposition. This cytoprotective effect of DXS was still present when the challenge with normal human serum was performed up to 48 hr after DXS treatment of the cells. DXS incubation of porcine ECs with and without prior tumor necrosis factor-alpha stimulation reduced xenogeneic cytotoxicity mediated by human NK cells by 47.3% and 25.3%, respectively. CONCLUSIONS: DXS binds to porcine cells and protects them from complement- and NK cell-mediated injury in vitro. It might therefore be used as a novel therapeutic strategy to prevent xenograft rejection and has potential for clinical application as an "EC protectant."

Mast cells in ongoing acute rejection: increase in number and expression of a different phenotype in rat heart transplants.

BACKGROUND: Mast cells (MC) are resident in healthy hearts and play important physiological and pathophysiological roles. In the transplanted heart, correlations have been found between MC number and the severity of rejection episodes, the intensity of chronic inflammation, and allograft arteriosclerotic changes. However, not much emphasis has been placed on the fact that resident donor MC, and infiltrating recipient MC do not forcedly need to share the same properties and function. To gain insight in the role of cardiac MC during acute, and ongoing acute rejection of heart transplants, we investigated MC kinetics and MC phenotype in a rat heart transplantation model. METHODS: Donor hearts from female Brown-Norway rats were transplanted to male Lewis rats. Immunosuppression was started at day 5 using ciclosporin and prednisolone. Connective tissue type MC (CTMC) were distinguished from mucosa type MC (MMC) by immunohistochemistry for rat MC protease (RMCP) -1 and -2. Expression of RMCP-1 and -2 mRNA was quantified by real time reverse transcription-polymerase chain reaction. Infiltrating Y chromosome positive MC were detected by fluorescence in situ hybridization. mRNA expression of interleukin-3 (IL-3) and of the two differentially spliced isoforms of kit ligand (KL, stem cell factor) was quantified using reverse transcription-polymerase chain reaction. RESULTS: Resident cardiac donor MC are almost exclusively CTMC and decrease in number during acute rejection. MC increase in number, and recipient MC invade the cardiac allograft during ongoing acute rejection. The phenotype of the invading MC is characterized by the expression of RMCP-2, or both RMCP-1 and RMCP-2, and thus resemble a MMC type. IL-3 mRNA is highly expressed, and the ratio of the differentially spliced mRNAs for KL-1 and KL-2 rises up to 2-fold during ongoing acute rejection. CONCLUSIONS: Our data show that MC in posttransplant hearts during ongoing acute rejection differ from MC in healthy hearts and isografts by expressing a different phenotype. Changes in IL-3 and KL expression might be responsible for the predominance of MMC over CTMC. The notion is of importance that MC in cardiac allografts may have properties and functions that differ from those in nontransplanted healthy hearts.