Andres L. Brodsky

Genetic variants in C5 and poor response to eculizumab.

BACKGROUND Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear. METHODS We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients. RESULTS Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab. CONCLUSIONS The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).

Forskolin induces U937 cell line differentiation as a result of a sustained cAMP elevation

The present study examines the effects of forskolin on U937 cell differentiation. We recently reported that dibutyryl cAMP (dbcAMP), but not cAMP-elevating agents such as histamine, promotes U937 cell differentiation. cAMP production elicited by stimulation of histamine H2 receptors showed a rapid, homologous desensitization, which might explain the dissimilar responses to histamine and dbcAMP. Forskolin induced an increase in cAMP levels in a concentration-dependent manner (EC50=30 microM) for an extended period of at least 24 h. Forskolin but not histamine (up to 100 microM), also inhibited cell growth in a dose-dependent fashion (EC50=22 microM). After 3 days of incubation, 75 microM forskolin induced U937 cell differentiation as judged by an increased rate of reduction of nitrobluetetrazolium (mean+/-S.E.M.: 21.3+/-6.6% in treated cells vs. 3.2+/-1.9% in the control group, P < 0.001) and an augmented chemotactic response to complement 5a (C5a) (33.2+/-5.9% in forskolin-treated vs. 0.34+/-0.12% in control cells, P < 0.01). Furthermore, c-Myc levels decreased following forskolin treatment, while the histamine H2 receptor agonist dimaprit had no effect. We conclude that forskolin induces U937 cell differentiation through a sustained rise in cAMP levels.

Rapid desensitization and slow recovery of the cyclic AMP response mediated by histamine H2 receptors in the U937 cell line

The present study focused on the desensitization process of the H(2) receptor in U937 cells and the recovery of the cyclic AMP (cAMP) response. Treatment of U937 leukemic cells with the H(2) histamine receptor agonists (+/-)-N(1)-[3-(3, 4-difluorophenyl)-3-(pyridin-2-yl)propyl]-N(2)-[3-(1H-imidazol-4-yl)p ropyl]guanidine (BU-E-75) and amthamine produced a rapid desensitization characterized by decreased cAMP production (T(1/2) = 20 min). Pretreatment with 10 microM BU-E-75 did not induce modifications in the responses to prostaglandin E(2), isoproterenol, or forskolin. H(2) receptor desensitization was not affected by protein kinase A and C inhibitors, but was reduced drastically by Zn(2+) and heparin, known to act as inhibitors of G protein-coupled receptor kinases. Recovery studies of the cAMP response showed that cAMP levels reached 50% of the initial values within 5 hr. Furthermore, desensitization produced an important decrease in the basal level of this cyclic nucleotide. The minimal value was observed 12 hr later, and corresponded to approximately 1.3% of the initial basal level (7.5 vs 0.1 pmol/10(6) cells). This result could be explained by an increase in phosphodiesterase activity following 10 microM BU-E-75 treatment. When cells were exposed for 2 hr to an H(2) agonist, binding assays showed no modification in the number of H(2) receptors; internalization began just after 8 hr. Although the initial desensitization seems to involve G protein-coupled receptor kinases, results indicate that additional mechanisms of regulation were triggered by the H(2) agonists.

Stevens‐Johnson Syndrome, Respiratory Distress and Acute Renal Failure Due to Synergic Bleomycin‐Cisplatin Toxicity

A patient presenting with a cervix uteri epidermoid carcinoma had acute renal failure after treatment with cisplatin and bleomycin. She later developed fatal bleomycin‐induced pneumonitis and Stevens‐Johnson syndrome. This is a very rare association of adverse effects. The risk of causing a drug interaction thus increasing the severity of bleomycin undesired effects must be taken into account when a nephrotoxic drug is added to the former. The authors stress the importance of monitoring renal function under these conditions.