Late gestation diet supplementation of resin acid-enriched composition increases sow colostrum immunoglobulin G content, piglet colostrum intake and improve sow gut microbiota.

Resin acid-enriched composition (RAC) mainly containing tall oil fatty acid with an active component of resin acid (RA) can improve the microbial population in the digestive system, change the microbial fermentation, and improve the feed conversion ratio. We investigated the effects of dietary supplementation of RAC on sow colostrum yield (CY), colostrum composition and gut microbiota. Tall oil fatty acid and RA are commonly termed RAC and CLA, pinolenic, abietic, dehydrobiotic acids are characteristic components of RAC. The experiment was conducted in three trials in three respective herds. Sows were fed with a control diet and the same diet supplemented with 5 g RAC/day per sow during the last week of gestation. The 16S ribosomal RNA gene sequencing technique was used to assess sows' faecal microbiota populations at farrowing. Colostrum nutritional composition, acute phase proteins (APPs) and immunoglobulin (Ig) content were also assessed. Individual piglets were weighed at birth and 24 h after the birth of first piglets in order to calculate CY and later at 3 to 4 weeks to calculate average daily gain. The RAC-fed sows had significantly higher IgG levels (P0.05), but those fed RAC had higher levels of colostrum serum amyloid A. Colostrum yield was significantly higher in RAC-fed sows in herds 2 and 3 with heavier piglets between 3 and 4 weeks of age (P0.05). Resin acid-enriched composition supplementation significantly increased some beneficial and fermentative bacteria (Romboutsia and Clostridium sensu stricto) than the control diet (P<0.01) while some opportunistic pathogens (Barnesiella, Sporobacter, Intestinimonas and Campylobacter), including Proteobacteria, were suppressed. Therefore, RAC added to the sow diet at late pregnancy increases colostrum IgG, colostrum availability for neonate piglets, and seems to promote better maternal intestinal microbial sources.

Validation of Brix refractometer to estimate colostrum immunoglobulin G content and composition in the sow.

Colostrum is an essential source of immunoglobulin G (IgG) for neonate piglets. However, colostrum IgG content and nutritional composition can vary considerably among sows due to age, parity, feeding regime and immunological background. Currently, there is no practical way to obtain information about colostrum IgG concentration at herd level. We evaluated sows' colostrum IgG content on-farm using a Brix refractometer and its performance was compared with that of an IgG ELISA. In addition, nutritional compositions of the colostrum samples were analyzed using Fourier transform IR spectroscopy. Colostrum samples (5 to 6 ml) (n=153) were obtained within 0 to 3 h of farrowing. However, to obtain a 24 h IgG profile for 11 sows, colostrum samples were collected at 0, 2, 4, 6, 8, 10, 16 and 24 h after farrowing. A 0.3 ml of freshly drawn colostrum sample was used for the on-farm measurement of Brix percentages using a digital refractometer shortly after collection. The remaining fractions of the samples were frozen and submitted to laboratory analysis for total IgG, using a commercially available pig IgG ELISA kit. For nutritional composition analysis, a 35 ml colostrum sample (n=34) was obtained immediately after birth of first piglet from the first three pairs of frontal teats. Colostrum concentrations of IgG averaged 52.03±30.70 mg/ml (mean±SEM) at 0 to 3 h after farrowing. Concentration of IgG decreased on average by 50% during the 1st day of lactation (P30%. Colostrum IgG concentration is highly variable among sows, Brix measurement of a sows' fresh colostrum is an inexpensive, rapid and satisfactorily accurate method of estimating IgG concentration, providing indication of differentiation between good and poor IgG content of colostrum.

Release of endogenous anti-inflammatory complement regulators FHL-1 and factor H protects synovial fibroblasts during rheumatoid arthritis.

Rheumatoid arthritis is a chronic inflammatory disease of unknown aetiology predominantly affecting cells and tissues of synovial joints. Here we show that the two important complement regulators FHL-1 and factor H play a protective anti-inflammatory role in rheumatoid arthritis. Expression analyses at the mRNA- and protein level show in vitro expression and secretion of both regulators by synovial fibroblasts derived from patients with rheumatoid arthritis. Similarly the two regulators are synthesized in vivo in diseased synovial tissue, and in particular synovial lining cells express high levels of FHL-1. The anti-inflammatory role of these regulators in rheumatoid arthritis is highlighted by their induction with IFN-gamma and dexamethasone, whilst the pro-inflammatory cytokine TNF-alpha had no effect. Transient transfection experiments with various FHL-1/factor H promoter-luciferase reporter constructs into cells of distinct origin show independent cell and tissue specific promoter regulated transcription of these two regulators. The inducible expression, specifically of FHL-1 has physiological consequences. By binding directly to surfaces the released proteins protect cells from inflammatory damage and complement-mediated cell lysis. This study shows a novel protective and anti-inflammatory role of the two important complement regulators FHL-1 and factor H in rheumatoid arthritis and suggests a disease controlling role of the two proteins.

Secretion of soluble complement inhibitors factor H and factor H-like protein (FHL-1) by ovarian tumour cells.

We observed that the soluble complement regulators factor H and factor H-like protein were abundantly present in ascites samples as well as in primary tumours of patients with ovarian cancer. RT-PCR and immunoblotting analyses showed that the two complement inhibitors were constitutively produced by the ovarian tumour cell lines SK-OV-3 and Caov-3, but not PA-1 or SW626 cells. The amounts of factor H-like protein secreted were equal to those of factor H. This is exceptional, because e.g. in normal human serum the concentration of factor H-like protein is below 1/10th of that of factor H. In ascites samples the mean level of factor H-like protein (130+/-55 microg ml(-1)) was 5.5-fold higher than in normal human serum (24+/-3 microg ml(-1)). Ovarian tumour cells thus preferentially synthesise factor H-like protein, the alternatively spliced short variant of factor H. The tumour cells were found to bind both (125)I-labelled factor H and recombinant factor H-like protein to their surfaces. Surprisingly, the culture supernatants of all of the ovarian tumour cell lines studied, including those of PA-1 and SW626 that did not produce factor H/factor H-like protein, promoted factor I-mediated cleavage of C3b to inactive iC3b. Subsequently, the PA-1 and SW626 cell lines were found to secrete a soluble form of the membrane cofactor protein (CD46). Thus, our studies reveal two novel complement resistance mechanisms of ovarian tumour cells: (i) production of factor H-like protein and factor H and (ii) secretion of soluble membrane cofactor protein. Secretion of soluble complement inhibitors could protect ovarian tumour cells against humoral immune attack and pose an obstacle for therapy with monoclonal antibodies.

Rituximab (anti-CD20) therapy of B-cell lymphomas: direct complement killing is superior to cellular effector mechanisms.

Rituximab (IDEC-C2B8, Mabthera(R)) is a chimeric (human-mouse) monoclonal antibody (MoAb) against the B-cell specific CD20-antigen. It has been used for the clinical treatment of non-Hodgkin's lymphomas, but variable clinical results suggest that some lymphoma cells remain resistant. In the present study we have evaluated the relative efficiencies of humoral and cell-mediated effector mechanisms complement-dependent cytotoxicity (CDC), antibody-(ADCC), complement-(CDCC) dependent cellular cytotoxicity and apoptosis on lymphoma cell killing by rituximab. Rituximab activated the cytolytic complement (C) cascade and induced a strong CDC, but the rituximab-triggered ADCC and CDCC were relatively ineffective. The CDC was strongly enhanced by antibodies against the C inhibitor CD59 (protectin). Neutralization of CD55 (DAF) and CD46 (MCP) had a similar but weaker effect. Rituximab also induced apoptosis but in a cell line-dependent fashion. The results strongly emphasize the role of direct CDC as the major, fast and efficient effector mechanism of rituximab. In the immunotherapeutic treatment of B-cell lymphomas, it is important to consider the role of C-regulatory proteins as an escape mechanism of the malignant cells. Our results suggest that the effect of rituximab therapy could be enhanced by combining it with neutralization of CD59.

Exceptional resistance of human H2 glioblastoma cells to complement-mediated killing by expression and utilization of factor H and factor H-like protein 1.

Of over 20 nucleated cell lines we have examined to date, human H2 glioblastoma cells have turned out to be the most resistant to complement-mediated cytolysis in vitro. H2 cells expressed strongly the membrane attack complex inhibitor protectin (CD59), moderately CD46 (membrane cofactor protein) and CD55 (decay-accelerating factor), but no CD35 (complement receptor 1). When treated with a polyclonal anti-H2 Ab, anti-CD59 mAb, and normal human serum, only 5% of H2 cells became killed. Under the same conditions, 70% of endothelial-like EA.hy 926 cells and 40% of U251 control glioma cells were killed. A combined neutralization of CD46, CD55, and CD59 increased H2 lysis only minimally, demonstrating that these complement regulators are not enough to account for the resistance of H2 cells. After treatment with Abs and serum, less C5b-9 was deposited on H2 than on U251 and EA.hy 926 cell lines. A reason for the exceptional resistance of H2 cells was revealed when RT-PCR and protein biochemical methods showed that the H2 cells, unlike the other cell lines tested, actively produced the soluble complement inhibitors factor H and factor H-like protein 1. H2 cells were also capable of binding human factor H from the fluid phase to their cell surface and promoted the cleavage of C3b to its inactive form iC3b more efficiently than U251 and EA.hy 926 cells. In accordance, anti-factor H mAbs enhanced killing of H2 glioblastoma cells. Taken together, our results show that production and binding of factor H and factor H-like protein 1 is a novel mechanism that these malignant cells utilize to escape complement-mediated killing.

Up-regulated expression of decay-accelerating factor (CD55) confers increased complement resistance to sprouting neural cells.

We studied gene expression in relation to induced neural differentiation in a human neural crest-derived cell line, Paju. Messenger RNA isolated before and after treatment with phorbol 12-myristate 13-acetate was analyzed by differential display reverse transcription PCR. A strongly up-regulated expression of decay-accelerating factor (DAF, CD55) was found to parallel the induced neural sprouting while the expression of two other complement regulatory proteins (CD59/protectin, CD46/membrane cofactor protein) remained unaltered during neural differentiation. The increased membrane expression of DAF, which was also seen on neural processes and growth cones, conferred elevated resistance to complement-mediated lysis. Our findings suggest that in sprouting neurons DAF expression is up-regulated to provide additional complement resistance to pathfinding axons/dendrites invading new environment. It is also suggested that membrane expression of DAF may constitute a marker of growing and regenerating neurons.

Resistance of ovarian teratocarcinoma cell spheroids to complement-mediated lysis.

We have shown previously that it is possible to target complement-mediated killing against cultured ovarian tumour cells in vitro. As malignant ovarian cells usually grow in solid nodules in vivo, we have in the present study examined the effectiveness of complement killing against ovarian teratocarcinoma cells (PA-1) growing in three-dimensional tumour microspheroids (TMSs). Our study shows that PA-1 cells growing in TMSs are less susceptible to complement-mediated killing than cells growing in monolayer cultures, even after neutralization of protectin (CD59), the main inhibitor of complement lysis. Cells in suspension and cells growing in TMSs showed a similar expression of membrane co-factor protein (MCP, CD46) and CD59. Decay-accelerating factor (DAF, CD55) was not detected on the surface of cells in suspension, but appeared focally on the outermost cell layers of the TMSs. Complement-activating antibodies bound to all PA-1 cells in suspension but only to the most peripherally located cells in TMSs, even though the target antigens were similarly expressed in the two systems. Antibody-induced complement activation on PA-1 cells in suspension led to C3 and C5b-9 deposition on most cells, while C3 and C5b-9 were only found on the outermost layers of the TMSs. The increased complement resistance of tumour cells growing in three-dimensional spheroids is partly because of an insufficient penetration of antibodies and complement into the TMSs. TMSs are a useful model for the development of more efficient ways to kill malignant cells in micrometastases with monoclonal antibodies and complement.

Expression of complement membrane regulators membrane cofactor protein (CD46), decay accelerating factor (CD55), and protectin (CD59) in human malignant gliomas.

Gliomas are malignant brain tumors, which, despite recent progress in surgical and radiological treatment, still have a poor prognosis. Since gliomas apparently resist immunological clearance mechanisms, we became interested in examining bow gliomas resist killing by the human complement system. The resistance of human cells to complement-mediated damage is, in large part, mediated by specific inhibitors of complement:membrane cofactor protein (CD46), decay-accelerating factor (CD55), and protectin (CD59). In the present study we examined the expression of complement regulators in 14 human glioma tumors and in 7 glioma cell lines (U251, U87, HS683, U373, U138, U118, and H2). Protectin was found to be strongly expressed by all glioma tumors and cell lines. Northern blotting analysis demonstrated the typical pattern of four to five protectin mRNAs in the glioma cells. Except for blood vessels, the expression of decay-accelerating factor was weak or absent in the tumors in situ, whereas in the cell lines its expression varied, ranging from negative to intermediate. Membrane cofactor protein was moderately expressed by all the cell lines but only weakly in the tumors. Cell-killing experiments demonstrated that the glioma cell lines were exceptionally resistant to C-mediated lysis. Five of the seven cell lines (U373, HS683, U118, U138, and H2) resisted complement lysis under conditions where most other cell lines were sensitive to killing. Neutralization experiments using specific monoclonal antibodies indicated that protectin was functionally the most important complement regulator in the glioma cells. The killing of the U87 and U251 cells could be significantly increased by a blocking anti-protectin monoclonal antibody, whereas for the other cell lines only moderate or no response was observed. The H2 cell line resisted killing by all antibodies and by complement. These results show that protectin is the most important complement regulator on human glioma cells. The exceptional complement resistance of some glioma cell lines suggests that they may utilize other, hitherto less well characterized, mechanisms to resist complement killing.

Targeted neutralization of the complement membrane attack complex inhibitor CD59 on the surface of human melanoma cells.

Major problems in the immunotherapy of human tumors with complement-activating monoclonal antibodies (mAb) are (i) inherent resistance of tumor cells to complement cytolysis and (ii) a possible undiscriminatory attack against normal cells. In the present study we have developed a procedure to simultaneously direct the complement membrane attack complex and neutralize its inhibitor CD59 (protectin) on human melanoma cells in vitro. G361 melanoma cells were selectively recognized in heterogenous cell mixtures by a complement-fixing mAb (R24) against the tumor cell GD3-ganglioside. Biotinylated anti-CD59 mAb (YTH53.1) was directed to the tumor cells with a high-affinity biotin-avidin bridge using a proportion of R24 as a biotinylated targeting mAb and avidin as a linker. Biotinylated anti-CD59 mAb lost its ability to activate complement, but retained its CD59-neutralizing activity. Thus, it was possible to avoid nonspecific lysis of surrounding erythrocytes and endothelial cells and direct the CD59-neutralizing effect to the tumor cells. As a result the tumor cells were efficiently killed by R24 plus complement while the bystander cells remained viable. These results suggest that it is possible to target an unrestricted complement membrane attack against GD3- and CD59-positive melanoma cells.