Rat polymeric IgA binds C1q, but does not activate C1.

Immune complexes, prepared with monoclonal rat IgA antibodies directed against DNP, activate the alternative pathway of the complement system in rat serum. In this study, the interaction of these monoclonal IgA antibodies with the classical pathway of complement was investigated. Monoclonal polymeric IgA (p-IgA) was shown to inhibit the IgG2b-mediated classical pathway-dependent lysis of TNP-coated sheep red blood cells. In addition, the binding of C3 to solid phase IgG2b immune complexes was inhibited by p-IgA. Monoclonal monomeric IgA (m-IgA) was much less efficient in this respect. To further analyse the effect of p-IgA on the activation of the classical pathway by IgG2b immune complexes, the interaction of p-IgA with C1 was studied. It was found that p-IgA antibodies bind C1q. No species-specificity was observed, since both rat and human C1q were bound. Whereas binding of C1q in C1 to IgG2b resulted in activation of C1, binding to p-IgA did not. The binding of C1q to both p-IgA and IgG2b could be inhibited by monoclonal antibodies directed against the globular heads of C1q, but not by monoclonal antibodies directed against the collagen tail. The formation of insoluble p-IgA immune complexes was inhibited in the presence of rat serum or C1. These studies indicate that C1q binds to p-IgA by its globular heads, and thereby may modulate classical pathway-mediated reactions such as the inhibition of immune precipitate formation.

Clearance kinetics and tissue distribution of aggregated human serum IgA in rats.

In the present study the clearance kinetics and tissue distribution of human polyclonal heat-aggregated serum IgA (AIgA) of different sizes in rats was studied after intravenous administration of 125I-AIgA. The 125I-AIgA of different sizes disappeared from the circulation in a biphasic manner with an initial rapid half-life (T1/2) and a second slower T1/2. The first T1/2 was related to the size of the 125I-AIgA: high molecular weight (MW) 125I-AIgA was cleared much faster than 125I-AIgA with a low MW. Relatively more degradation products were observed in blood when high MW 125I-AIgA were injected as compared to low MW 125I-AIgA. The AIgA were mainly taken up by the liver. Eight minutes after injection of high MW 125I-AIgA, 90% of the injected dose was found in the liver, whereas less than 2% was detected in the spleen. Very little activity was detectable in other organs, such as lungs, heart and kidneys. In the present study 1-3% of the injected 125I-AIgA were found in the bile. Analysis of this material revealed that low MW 125I-AIgA were transported more efficiently to the bile than high MW 125I-AIgA. To obtain more insight into the receptors involved in the clearance of 125I-AIgA, rats were pretreated with ovalbumin or asialofetuin. The clearance of 125I-AIgA of different sizes was inhibited when rats were pretreated with asialofetuin. Pretreatment with ovalbumin had no effect on the clearance rates of 125I-AIgA. These results suggest a role for carbohydrate receptors, which recognize glycoprotein-containing galactose terminal residues on Kupffer cells, in the clearance of 125I-AIgA.

Activation of complement by human serum IgA, secretory IgA and IgA1 fragments.

UNLABELLED: Activation of the complement (C) system by human IgA was studied. Both subclasses of IgA, IgA1 and IgA2, and secretory IgA were shown to activate C, as determined by deposition of C3 on glutaraldehyde-activated microwells coated with IgA. The activation of the C system occurred in the presence of MgEGTA and not in D-deficient serum. In addition to C3, deposition of properdin (P) but not of C4 was detected. These results indicate that C activation, as determined by measuring deposition of C3 and P, occurred by the alternative pathway (AP). The data further show that the major part of the hinge region, which is deleted in IgA2 as compared with IgA1 and which forms the major structural difference between the two subclasses, is not involved in C activation. Reduction and alkylation destroyed the ability of IgA to activate C, as has also been demonstrated for IgG. In order to define the C activating region of the IgA molecule, several fragments of IgA1 were tested. The four-chain molecules F(ab')2 and F(abc)2 were shown to activate the AP. No activation was observed with the two-chain fragments Fab and Fc. The Fc fragment of IgA also did not activate the CP, as does the Fc fragment of IgG. This indicates that activation of the AP of C by IgA is dependent on the presence of the F(ab')2 fragment. IN CONCLUSION: human IgA does activate C by the AP. This activation requires an intact F(ab')2 fragment.

The pharmacokinetic plasma profile of mitomycin C, measured after sequential intermittent intravenous administration.

The pharmacokinetic plasma profile of mitomycin C (MMC) was studied during sequential courses in man. MMC was given repeatedly as i.v. bolus injections at fixed dose levels to the same patient either as a single agent or as part of different combination chemotherapy regimens. Large interindividual variations between the various pharmacokinetic parameters were observed. Statistical analysis showed no significant differences between average pharmacokinetic parameters when comparing the first and the second MMC injection, except for the total body clearance (Cltot). The Cltot was higher for the second injection when compared to the first injection in a group of patients who received MMC as a single agent (10 patients). For a group of patients receiving MMC as part of a combination therapy the average values of Cltot of the first when compared to the second injection were not statistically different (nine patients). This observation could not be correlated with clinical observations on toxicities.

Mitomycin C-induced organ toxicity in Wistar rats: a study with special focus on the kidney.

Two studies were performed to investigate acute and chronic organ toxicity after Mitomycin C (MMC) administration in Wistar rats. Six rats received 2.5 mg/kg MMC i.p. once and were followed for 5 consecutive days. The alanine aminopeptidase (AAP)/creatinine ratio increased significantly, compared to a control group receiving saline. Four groups of rats were injected i.p. weekly for 5 weeks; 6 control rats with saline, 7 rats with 1.7 mg/kg of MMC, 7 rats with 10 mg/kg 5-fluorouracil (5-FU) and 7 rats with MMC as well as 5-FU. The latter two groups were included to study possible toxicity synergism between the two drugs. A significant decrease in AAP excretion in the MMC group, as well as a nonsignificant decrease in the MMC/5-FU group were the most remarkable observations. Light microscopy did not show renal changes, but did not show alveolar septal congestion after repeated MMC injections. It is concluded that MMC causes tubular damage in Wistar rats, with acute leakage of enzyme from the cells, followed by enzyme depletion during chronic treatment. Also MMC induces pulmonary changes in Wistar rats. To what extent these changes represent early stages of toxicity remains to be elucidated.

Pharmacokinetics and toxicity of mitomycin C in rodents, given alone, in combination, or after induction of microsomal drug metabolism.

The pharmacokinetics of mitomycin (MMC) was studied in Wistar rats. Up to five half-lives, the plasma concentration-time curve was biphasic. The AUC changed linearly with increasing doses between 0.5 and 7.5 mg/kg, which corresponds to 0.2 and 3 times the LD50 value in rats. Most of the drug was metabolized, and only 1%-2% and 10%-15% of the dose was eliminated unchanged by biliary and urinary excretion, respectively. The AUC of MMC at the LD50 is slightly less than that reported for the human MTD. Inoculation of MMC together with 5-fluorouracil and doxorubicin did not change the terminal half-life of MMC but decreased the total body clearance and the volume of distribution. The lack of significant influence of phenobarbital and 3-methylcholanthrene pretreatment on the terminal elimination half-life suggests that microsomal drug-metabolizing enzymes inducible by these compounds do not play a decisive role in the in vivo biotransformation of MMC.

The IgA-binding lectin jacalin induces complement activation by inhibition of C-1-inactivator function.

Jacalin, a D-galactose-specific lectin from jackfruit, interacts with human IgA and one or two other serum proteins. Incubation of jacalin with fresh human serum was shown to result in activation of the complement system. Therefore the mechanism of complement activation by jacalin was studied. Jacalin was extracted from jackfruit seeds (crude preparation) and purified to homogeneity by affinity chromatography on IgA-Sepharose to yield a pure preparation of jacalin. Both crude and pure jacalin were able to activate complement, accompanied by conversion of C3. Consumption of C1, C4 and C-1-inactivator (C-1-In) indicated involvement of the classical pathway. Aggregated IgG (AIgG) caused partial (38%) and jacalin induced complete consumption of C1-In functional activity. It was found upon Ouchterlony analysis that jacalin forms a precipitation line with purified C-1-In. In addition binding of 125I-C-1-In to jacalin-Sepharose was observed, and this binding was inhibitable by either secretory IgA or D-galactose. Next to binding of jacalin to C-1-In, jacalin was also shown to inhibit the functional activity of C-1-In. These results indicate that jacalin induces complement activation by inhibition of C-1-In function and thereby facilitates the activation of precursor C1 in either the absence or presence of low amounts of C1 activators.

Activation of the alternative pathway of complement by human serum IgA.

In order to study the activation of complement by soluble aggregates of human polyclonal serum IgA, lysis of sheep erythrocytes (E) coated with several IgA preparations was used as a model. A complement nonactivating monoclonal mouse IgG1 against IgA was used to coat the cells. IgA, isolated from normal human serum, was aggregated by either N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), glutaraldehyde, carbodiimide or heating. Depending on the size of the aggregates, and on the method of aggregation, E coated with aggregated IgA (E gamma 1.AIgA) could be lysed. The alternative pathway of complement appeared to mediate the lysis because the latter was observed in the presence of EGTA containing 5 mM Mg2+ (MgEGTA) and properdin (P) was deposited on the cells. Furthermore, no lysis was observed in C3-deficient serum. In the absence of AIgA the cells were not lysed, and no P deposition was observed. In another set of experiments E gamma 1.AIgA were first reacted with purified C3, B, D and P for 30 min at 30 degrees C, and subsequently in rat serum EDTA at 37 degrees C. Lysis occurred when E gamma 1.AIgA were prepared using SPDP-, glutaraldehyde- or carbodiimide-AIgA. Incubation of 100 micrograms/ml SPDP-AIgA with normal human serum for 30 min at 37 degrees C in the presence or absence of MgEGTA also induced consumption of total complement. The other soluble AIgA preparations were less effective in activating complement. These results suggest that polymeric serum IgA is capable of activating the alternative pathway of complement.

Absence of interaction between furosemide and mitomycin C.

The possible interaction between furosemide and mitomycin C (MMC) was studied in five patients. The pharmacokinetics of MMC were studied using an HPLC assay. Furosemide was administered prior to, or 120 min after MMC. Furosemide did not change the pharmacokinetics of MMC, nor did it change the amount of MMC excreted in the urine. There appears to be no interaction between the two drugs.

Relationship between clinical parameters and pharmacokinetics of mitomycin C.

Although the number of reports on mitomycin C (MMC) pharmacokinetics is increasing, data on possible relations between clinical parameters and pharmacokinetics are usually lacking. The present report concerns the results of a detailed study on this subject in 35 patients receiving MMC, either as a single agent or as a part of combination chemotherapy. MMC concentrations were determined by HPLC. T1/2 beta varied from 23 to 78 min, VD from 11 to 48 l/m2, Cl tot from 12 to 42 l/h per m2, and AUC from 138 to 1221 micrograms/h per l, confirming previously reported data. Infusion time, cholestasis, and urinary pH did not influence the pharmacokinetic data. There were no relations between other clinical data and pharmacokinetics, nor between AUC and bone marrow toxicity. An interaction between MMC and furosemide could not be excluded, but there was no interaction with other comedication. Consecutive pharmacokinetics in 6 patients showed consistent results. Because renal impairment does not alter MMC pharmacokinetics and renal excretion is not a major route of elimination, it is suggested that renal impairment does not call for dose adjustment.

The difference in pharmacokinetics of mitomycin C, given either as a single agent or as a part of combination chemotherapy.

In a previous report it was suggested that the total body clearance of mitomycin C (MMC) was different after single agent treatment compared to combination chemotherapy. This suggestion was based on recalculations to one dose level. In the present study a fixed dose of 10 mg/m2 was used. Seven patients on single agent MMC and eight on combination chemotherapy were studied. Terminal half-life varied from 25 to 78 mm, volume of distribution from 7 to 73 l/m2, total body clearance from 11 to 56 l/h per m2, and area under the plasma concentration-time curve (AUC) from 177 to 933 micrograms/h per l. Total body clearance was significantly higher and AUC significantly lower in patients on combination chemotherapy. The cause of this difference was not investigated.