Pediatric Heart Transplant Rejection After COVID-19 Infection.

BACKGROUND: Infections have been associated with rejection episodes in solid organ transplant recipients. We report an association between COVID-19 infection and heart transplant (HT) rejection. CASE DESCRIPTION: The patient was 14 years old and 6.5 years post-HT. He developed symptoms of rejection within 2 weeks of COVID exposure and presumed infection. CONCLUSIONS: In this case, COVID-19 infection closely preceded significant rejection and graft dysfunction. Further study is needed to establish a correlation between COVID-19 infection and rejection in HT patients.

Interactions between CYP2E1 and CYP2B4: effects on affinity for NADPH-cytochrome P450 reductase and substrate metabolism.

Studies in microsomal and reconstituted systems have shown that the presence of one cytochrome P450 isoform can significantly influence the catalytic activity of another isoform. In this study, we assessed whether CYP2E1 could influence the catalytic activity of CYP2B4 under steady-state turnover conditions. The results show that CYP2E1 inhibits CYP2B4-mediated metabolism of benzphetamine (BNZ) with a K(i) of 0.04 µM. However, CYP2B4 is not an inhibitor of CYP2E1-mediated p-nitrophenol hydroxylation. When these inhibition studies were performed with the artificial oxidant tert-butyl hydroperoxide, CYP2E1 did not significantly inhibit CYP2B4 activity. Determinations of the apparent K(M) and k(cat) of CYP2B4 for CPR in the presence of increasing concentrations of CYP2E1 revealed a mixed inhibition of CYP2B4 by CYP2E1. At low concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR increased up to 23-fold with virtually no change in the k(cat) for the reaction, however, at higher concentrations of CYP2E1, the apparent K(M) of CYP2B4 for CPR decreased to levels similar to those observed in the absence of CYP2E1 and the k(cat) also decreased by 11-fold. Additionally, CYP2E1 increased the apparent K(M) of CYP2B4 for BNZ by 8-fold and the apparent K(M) did not decrease to its original value when saturating concentrations of CPR were used. While the individual apparent K(M) values of CYP2B4 and CYP2E1 for CPR are similar, the apparent K(M) of CYP2E1 for CPR in the presence of CYP2B4 decreased significantly, thus suggesting that CYP2B4 enhances the affinity of CYP2E1 for CPR and this may allow CYP2E1 to out-compete CYP2B4 for CPR.

Uncovering the role of hydrophobic residues in cytochrome P450-cytochrome P450 reductase interactions.

Cytochrome P450 (CYP or P450)-mediated drug metabolism requires the interaction of P450s with their redox partner, cytochrome P450 reductase (CPR). In this work, we have investigated the role of P450 hydrophobic residues in complex formation with CPR and uncovered novel roles for the surface-exposed residues V267 and L270 of CYP2B4 in mediating CYP2B4--CPR interactions. Using a combination of fluorescence labeling and stopped-flow spectroscopy, we have investigated the basis for these interactions. Specifically, in order to study P450--CPR interactions, a single reactive cysteine was introduced in to a genetically engineered variant of CYP2B4 (C79SC152S) at each of seven strategically selected surface-exposed positions. Each of these cysteine residues was modified by reaction with fluorescein-5-maleimide (FM), and the CYP2B4-FM variants were then used to determine the K(d) of the complex by monitoring fluorescence enhancement in the presence of CPR. Furthermore, the intrinsic K(m) values of the CYP2B4 variants for CPR were measured, and stopped-flow spectroscopy was used to determine the intrinsic kinetics and the extent of reduction of the ferric P450 mutants to the ferrous P450--CO adduct by CPR. A comparison of the results from these three approaches reveals that the sites on P450 exhibiting the greatest changes in fluorescence intensity upon binding CPR are associated with the greatest increases in the K(m) values of the P450 variants for CPR and with the greatest decreases in the rates and extents of reduced P450--CO formation.