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Biomedical subjects

R K Stad

Publications and source records attributed to R K Stad.

17 recordsLinked to original sources
PubMed1995

Influence of Duraflo II heparin-treated extracorporeal circuits on the systemic inflammatory response in patients having coronary bypass.

Cardiopulmonary bypass generates a systemic inflammatory response, including the activation of leukocytes, contributing to postoperative morbidity. To evaluate whether the use of heparin-treated extracorporeal circuits could reduce the inflammatory reaction in patients undergoing cardiopulmonary bypass, we conducted a prospective clinical study on 14 patients having coronary artery bypass in whom perfusion was done randomly with either Duraflo II heparin-treated circuits or with nontreated circuits. In both groups systemic heparinization was performed before cardiopulmonary bypass. The use of heparin-treated circuits resulted in a reduction of systemic inflammatory activation during cardiopulmonary bypass. This was reflected by lower plasma levels of soluble tumor necrosis factor receptors (p < 0.05) and of interleukin-6 and interleukin-8 (p < 0.05), manifest after release of the aortic crossclamp. Furthermore, 6 and 12 hours after aortic crossclamp release significantly lower levels of the soluble E-selectin (p < 0.05) were observed in the Duraflo II group. In patients in whom noncoated circuits were used, a significant decrease in circulating soluble intercellular adhesion molecule 1 (p < 0.05) was found early during bypass. All these observations suggest that the use of a heparin-treated extracorporeal circuit reduces the systemic inflammatory activation and may after the leukocyte-endothelium interaction.

Cardiopulmonary Bypass↗
PubMed1995

The involvement of Kupffer cells and liver endothelial cells in the clearance of large sized soluble IgA aggregates in rats.

These findings suggests that AIgA is bound both by KC and EC in normal situations. Because of the great phagocytic capacity of KC, the contribution of EC in handling AIgA in normal rats is minimal. However, when KC are defect or absent (as after Cl2MDP treatment), the handling of AIgA by EC may become of mayor importance, i.e. it will take over the phagocytic function of KC. Studies concerning a possible receptor on EC involved in binding of AIgA are in progress.

Animals↗
PubMed1994

Differential regulatory effects of adenosine on cytokine release by activated human monocytes.

Adenosine is an endogenous nucleoside that can modulate the function of cells involved in the inflammatory response, such as polymorphonuclear leukocytes (PMN) and monocytes. Production and release of cytokines by activated mononuclear phagocytes is an important event in the pathogenesis of ischemia-reperfusion injury, a pathologic phenomenon that is associated with excessive ATP catabolism and subsequent local release of adenosine. The "retaliatory" metabolite adenosine has been shown to interfere with PMN function, thereby attenuating the deleterious consequences of ischemia and reperfusion. In this study, we demonstrate that adenosine inhibits the production of TNF-alpha, IL-6, and IL-8 by LPS-activated human monocytes with a differential potency. The A2 receptor-specific adenosine analogues 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine (NECA) were most effective in attenuating LPS-induced cytokine production, whereas the A1-selective adenosine analogue N6-cyclopentyladenosine (CPA) was less effective, indicating that inhibition of cytokine production by adenosine is primarily an A2 receptor-mediated event. The observed inhibitory effects were not restricted to endotoxin-induced cytokine production, because adenosine also inhibited TNF-alpha production by monocytes stimulated with the proinflammatory cytokine IL-1 beta. Again, 2-chloroadenosine and NECA reduced IL-beta-induced TNF-alpha production more potently than CPA. In contrast, adenosine enhanced production of IL-6 and IL-8 by monocytes stimulated with IL-1 beta. Furthermore, only 2-chloroadenosine, but not NECA, strongly inhibited cytokine-induced IL-6 and IL-8 production. These results suggest an additional A2 receptor-mediated mechanism of retaliatory action of adenosine under pathologic conditions where cytokine production by activated mononuclear phagocytes is involved, such as ischemia-reperfusion injury and septic shock.

Adenosine↗
PubMed1994

Hereditary C6 deficiency in a strain of PVG/c rats.

A chance observation has led to the discovery of a strain of PVG rats (PVG/c-) which are deficient in complement (C) component C6. Analysis of total haemolytic activity (CH50) of PVG/c- serum revealed an absent CH50 activity compared with serum of other rat strains and of a PVG/c rat (PVG/c+) that showed normal C activity. Thus, the PVG/c- rat was unable to activate the C5b-9 membrane attack complex. To gain insight into the complement abnormalities, analysis of individual C components was performed. Testing the PVG/c- serum in a C6 haemolytic assay and using deficient human sera showed a deficiency of C6 in the PVG/c- rat. Highly purified human C6 and human sera deficient in other components were able to reconstitute the CH50 activity of the PVG/c- rat. The possibility that an inactivator of C was present in PVG/c- serum was excluded. The deficiency was found to be inheritable and under the control of an autosomal recessive gene. Furthermore, tissue antigens and immunity of the PVG/c- rat were found to be identical to those determined in the PVG/c+ rat. With regard to their health status, the PVG/c- animals seem to have no disadvantages compared with PVG/c+ rats when held under the same conditions within the protected environment of animal facilities. Taken together, both rat strains provide an unique animal model for studying the biological role of C, particularly the C5b-9 membrane attack complex in experimental medicine.

Animals↗
PubMed1993

Role of liver endothelial and Kupffer cells in clearance of human C1q in rats.

In the present study the contribution of rat liver endothelial cells (EC) and Kupffer cells (KC) in the clearance of human (hu) C1q in rats was investigated. In untreated rats and rats depleted from KC the clearance kinetics and the tissue distribution of hu C1q were measured. In untreated rats, the clearance of hu C1q occurred in a monophasic manner with a half-life of 66 +/- 26.7 min. The clearance of hu C1q in KC-depleted rats was delayed significantly (p < 0.001) and occurred with a half-life of 217 +/- 78.8 min. Fifteen min after injection, 11 +/- 3.5% of hu C1q was found in the liver of untreated rats and 8 +/- 1.4% was found in the liver of KC-depleted rats. The percentage non-trichloroacetic acid precipitable activity in the circulation, as a measure for degradation of C1q, reached a level of 11.6 +/- 5.6% at 240 min in untreated rats compared with 4.6 +/- 5.8% in KC-depleted rats. Double immunofluorescence staining 5 min after administration of C1q in untreated rats, revealed that C1q was associated with KC and EC in the liver. Fifteen minutes after i.v. injection of hu C1q, there was an uptake of C1q in the hepatocytes. In KC-depleted rats, 5 min after administration of hu C1q, C1q was bound to the EC. Fifteen minutes after injection, C1q was also found in the hepatocytes. Electron microscopical studies revealed that C1q binds to EC, and that it is internalized in the hepatocytes and KC. The clearance of hu C1q in untreated rats was inhibited by preadministration of high concentrations of bovine C1q. These data show that rats depleted from KC are able to bind, internalize and degrade C1q, and that EC may play a role in the handling of C1q and C1q bound to immune complexes.

Animals↗
PubMed1993

An acute model for IgA-mediated glomerular inflammation in rats induced by monoclonal polymeric rat IgA antibodies.

An acute model for IgA-mediated glomerular inflammation in rats was induced by the in situ deposition of IgA directly into the glomerular mesangium. F(ab')2 anti-Thy1 MoAb was used to anchor an antigen, DNP (2,4-dinitrophenol), in the glomeruli of rats. Subsequent infusion of rat polymeric (p-) or monomeric (m-) IgA MoAb with specificity for DNP resulted in mesangial deposition of IgA in both groups of rats. However, acute proteinuria was observed only in p-IgA-treated rats and not in PBS- or m-IgA-treated rats. Immunofluorescence analysis revealed deposition of C3 in an identical pattern to that of IgA in the glomeruli of p-IgA-treated rats. No mesangial deposits of C4 or C1q were seen in these animals. Rats receiving m-IgA or PBS displayed no detectable C3, C4 or C1q deposition. The amount of proteinuria in p-IgA-treated rats was related to the amount of deposited C3. The presence of intraglomerular monocytes was only observed 2 days after p-IgA injection. By light microscopy, aneurysm formation, mesangial hypercellularity and matrix expansion were seen only in p-IgA-treated rats. However, by 37 days post-injection complete resolution of the lesions was observed. No histological renal changes were observed in PBS- or m-IgA-treated rats. In conclusion, an acute form of IgA-mediated nephritis in rats was induced by p-IgA but not by m-IgA. This reproducible model provides a basis for further study into the mechanisms of IgA-mediated glomerular inflammation.

2,4-Dinitrophenol↗
PubMed1992

In vivo activation of complement by IgA in a rat model.

In this study we investigated the capacity of rat IgA to activate complement (C) in vivo in a rat model. Rat monomeric (m-), dimeric (d-) and polymeric (p-) IgA MoAbs were injected intravenously and assessed for deposition of C3 and C4 on IgA. By ELISA it was shown that both d- and p-IgA bound C3 whereas no binding of C3 by m-IgA was observed. Polymeric IgA was more efficient in binding of C3 as compared with d-IgA. However, in haemolytic assays no consistent decrease of plasma complement levels was observed except for dimeric IgA which induced a marginal consumption of AP50. When rats were pre-treated with cobra venom factor (CVF) to deplete C3, no C3 deposition was found on m-, d- or p-IgA. Neither m- nor d- or p-IgA was able to bind C4 in vivo. In agreement with the results described above, large sized polymeric IgA was shown to be taken up by Kupffer cells (KC) together with C3. No C3 was detected when rats were depleted of C using CVF. Taken together, the experimental data suggest that d- and p-IgA are able to activate C via the alternative pathway in vivo.

Animals↗
PubMed1991

Complement enhances the clearance of large-sized soluble IgA aggregates in rats.

In the present study the involvement of the complement system (C) in the clearance of soluble IgA aggregates in the rat was studied. Monoclonal monomeric IgA (mIgA) antibody (which does not activate C) or aggregated polymeric IgA (aIgA; which activates C) were administered intravenously to phosphate-buffered saline-treated and complement-depleted [Cobra venom factor (CVF)-treated] rats and assessed for clearance from the circulation. In control rats, mIgA was cleared in a biphasic fashion with a first half-life (T1/2) of 29.5 +/- 14.2 min and a second T1/2 of 230 +/- 176 min. No differences were observed in clearance of mIgA in CVF-treated rats as compared to PBS-treated rats. In PBS-treated rats, aIgA with a size between 20 S and 150 S disappeared very rapidly from the circulation with a first T1/2 of 1.1 +/- 0.4 min and a second T1/2 of 23.2 +/- 11.3 min. In CVF-treated rats the clearance of aIgA was significantly delayed as compared to that in control rats, namely with a first T1/2 of 7.3 +/- 2.6 min and a second T1/2 of 64.2 +/- 19.4 min. Immunohistochemical studies of the liver (which is the main site of clearance of aIgA) revealed that Kupffer cells (KC) are mainly responsible for the uptake of aIgA. Furthermore, in PBS-treated rats aIgA deposition was accompanied by C3 deposition in the KC. In CVF-treated rats, the percentage of KC containing aIgA was significantly lower during the first 16 min after aIgA administration as compared to PBS treated rats. In addition no detectable C3 was found in KC of CVF-treated rats. These results indicate that KC play an important role in the clearance of large molecular weight IgA in rats and that C facilitates the clearance of these complexes from the circulation.

Animals↗
PubMed1991

Kupffer cell depletion in vivo results in clearance of large-sized IgA aggregates in rats by liver endothelial cells.

We investigated the clearance kinetics and tissue distribution of different sized IgA in normal and macrophage-depleted rats. Rats were injected iv with liposomes containing dichloromethylene diphosphonate (DMDP). DMDP treatment resulted in complete depletion of liver macrophages 24-48 h after administration. Normal and macrophage depleted rats were injected intravenously with monomeric, dimeric, polymeric or aggregated polymeric IgA (AIgA) and assessed for blood clearance and tissue distribution. In normal rats, clearance of IgA was size dependent, i.e. a faster clearance with increasing size. No differences in clearance kinetics were observed of the different sized IgA between normal and DMDP-treated rats. TCA non-precipitable radioactivity, a measure for degradation of IgA, was found in the circulation of normal and DMDP-treated rats after AIgA administration. The liver was the main organ responsible for the clearance of IgA in normal and DMDP-treated rats. Immunofluorescence studies on liver biopsies indicated that AIgA was associated with Kupffer cells in normal rats. Electron microscopical studies revealed that the AIgA was internalized and located in vesicles in Kupffer cells. In DMDP-treated rats the AIgA was associated with endothelial cells and electron microscopy studies showed that this AIgA was taken up by endothelial cells. These data show that rat liver endothelial cells are able to bind, internalize and degrade AIgA in situations where Kupffer cells are absent, and that these cells may play an important role in the handling of AIgA and IgA-immune complexes.

Alkaloids↗
PubMed1991

Kupffer cell depletion in vivo results in preferential elimination of IgG aggregates and immune complexes via specific Fc receptors on rat liver endothelial cells.

In the present study we have investigated the clearance kinetics and tissue distribution of monomeric (m) IgG and soluble aggregates of IgG (AIgG) and immune complexes (IC) in normal and Kupffer cell (KC) depleted rats. In normal rats, clearance of mIgG occurred in a biphasic manner with a first half-life (T1/2) (T1) of 36.3 +/- 6.3 min and a second T1/2 (T2) of 168.4 +/- 4.7 min. AIgG composed of 20-27 IgG molecules per aggregate were cleared significantly faster than mIgG with a T1 of 2.5 +/- 0.1 min and a T2 of 32.5 +/- 5.6 min. KC depletion did not have a significant effect on the clearance rate of mIgG (T1: 33.4 +/- 8.9 min; T2; 159.5 +/- 12.5 min), while clearance of AIgG was delayed significantly with T1 4.8 +/- 0.7 min and T2 41.2 +/- 3.2 min. Eight minutes after injection, 77% of AIgG was found in the liver in normal rats while 62% was found in the liver of KC-depleted rats. Double immunofluorescence studies indicated that AIgG in the liver was associated with KC and endothelial cells (EC) in normal rats. In KC-depleted rats, AIgG was strongly associated with EC. A similar staining pattern was observed when IgG-immune IC were administered. The clearance of AIgG in KC-depleted rats was inhibited fully by pre-administration of high concentrations of IgG but not by pretreatment with IgA. asialofetuin (ASFe) or ovalbumin (OVA). Aggregated F(ab')2IgG was cleared with a comparable rate to mIgG from the circulation, again suggesting Fc gamma receptor-mediated elimination of AIgG by EC. There was a reduced degradation of AIgG in rats depleted of KC as compared with normal rats. These data suggest binding and degradation of AIgG by EC in vivo.

Animals↗
PubMed1991

Deposition of IgA is associated with macrophage influx in the kidney of rats.

In the present study we treated rats with N-nitrosodimethylamine (DMN) or D-galactosamine (GALN) to achieve increased circulating IgA levels in rats. GALN-treated rats showed a six-fold increase in serum IgA levels after the first intraperitoneal (i.p.) injection, whereas a 10-fold increase after a second i.p. injection of GALN was seen. DMN-treated rats showed a three-fold increase in serum IgA levels. No differences were observed in IgG and IgM levels between treated and non-treated rats. Sequential renal biopsies analysed by immunofluroescence exhibited mesangial deposits of IgA with different intensities of C3 deposition. Rats treated with GALN showed more IgA deposition in the kidney than DMN-treated rats. The IgA deposition together with C3 was more prominent in rats treated with GALN than in rats treated with DMN. The deposition of C3 together with IgA was associated with an influx of monocytes as detected by ED-1, an antibody directed against a rat monocyte marker. These studies provide evidence that an increase in serum IgA levels is associated with deposition of IgA in the kidney and that IgA has an inflammatory potential.

Animals↗
PubMed1991

Immunoglobulin A: interaction with complement, phagocytic cells and endothelial cells.

Deposits of IgA together with complement (C) in different organs support the hypothesis that IgA can trigger inflammatory mechanisms. Some inflammatory mechanisms may be caused by activation of C and phagocytic cells. Therefore, it is essential to understand the interaction of IgA with C and phagocytic cells. Studies will be described demonstrating that polymeric human serum IgA is able to activate the alternative pathway of C and that the activating principle is located in the intact F(ab')2 portion of the molecule. Activation of C is dependent on the molecular composition of IgA, as derived from results obtained with rat monoclonal IgA antibodies. Furthermore, it is demonstrated that polymeric IgA (pIgA) and dimeric IgA (dIgA) are potent activators of C in a homologous rat model, whereas monomeric IgA (mIgA) has a very poor C-activating potential. The interaction of IgA with phagocytic cells induces phagocytosis and release of H2O2 by granulocytes, which may contribute to tissue damage. Little is known about the clearance mechanism of IgA. It is shown in this report that Kupffer cells and C play an important role in the clearance of IgA immune complexes (IC). Clearance of large-sized IgA IC occurs via different receptors present on Kupffer cells. Finally, a new aspect will be described: the interaction of IgA with endothelial cells. Rat liver endothelial cells are able to eliminate IgA IC from the circulation via specific receptors when no Kupffer cells are present. These observations may contribute to our knowledge on diseases such as IgA nephropathy and Henoch-Schönlein purpura. The studies summarized and presented here illustrate the inflammatory potential of IgA.

Animals↗
PubMed1988

Enrichment and selection of hybrid hybridomas by Percoll density gradient centrifugation and fluorescent-activated cell sorting.

Hybrid hybridomas, producing bi-specific monoclonal antibodies that react with horseradish peroxidase and human IgA1 were isolated by sorting the double-fluorescent cells on single-cell basis after fusion of two hybridomas, previously labelled green or red by octadecylamine-FITC or -TRITC, respectively. The double-fluorescent fused cells were significantly different in AXL (size) and RAS (internal structure) distribution compared with the (non-fused) mono-fluorescent cells. The percentage of double-fluorescent cells and the viability of these cells could be increased by Percoll density gradient centrifugation. As a result, there was an 8-fold increase of total isolated hybrid hybridomas (up to 30% of all tested clones) compared to isolations without Percoll density gradient centrifugation. All the isolated hybrid hybridoma clones had similar amounts of DNA, equal to the sum of the DNA of both parental hybridomas.

Antibodies, Monoclonal↗
PubMedDate not supplied

Complement depletion abolishes IgA-mediated glomerular inflammation in rats.

Recently, we developed an acute model for IgA-mediated glomerular inflammation in rats in which it was shown that polymeric (p) but not monomeric (m) IgA-containing immune complexes induce acute glomerular inflammation. The glomerular IgA-mediated inflammation is characterized by the activation of complement (C), the presence of intraglomerular macrophages and proteinuria. In the present study, we investigated the role of C in this IgA-mediated nephritis. Rats were pretreated either with cobra venom factor (CVF) to deplete them of circulating C3 or with phosphate-buffered saline followed by introduction of mesangial IgA deposits. Upon deposition of pIgA in the mesangial area, acute proteinuria was observed only in normocomplementemic rats and not in C-depleted animals. Immunofluorescent analysis revealed deposition of C3 and C9 in a pattern identical to that of IgA in the glomeruli of normal rats. Rats pretreated with CVF displayed clear mesangial deposition of IgA in the absence of C3 and C9. In none of the two groups were C4 deposits seen, indicating activation of C via the alternative pathway. In normocomplementemic animals, deposition of IgA together with C3 was associated with an influx of macrophages at day 2. C-depleted rats receiving pIgA also showed an influx of macrophages at 24 h following CVF administration and 1 and 2 days after IgA injection. However, no proteinuria was seen. To obtain insight into the mechanism of macrophage influx in the CVF-treated rats, we also analyzed the number of intraglomerular macrophages in rats receiving only CVF, without introduction of mesangial IgA deposits.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗