Current practices for treatment of respiratory syncytial virus and other non-influenza respiratory viruses in high-risk patient populations: a survey of institutions in the Midwestern Respiratory Virus Collaborative.

BACKGROUND: The optimal treatment for respiratory syncytial virus (RSV) infection in adult immunocompromised patients is unknown. We assessed the management of RSV and other non-influenza respiratory viruses in Midwestern transplant centers. METHODS: A survey assessing strategies for RSV and other non-influenza respiratory viral infections was sent to 13 centers. RESULTS: Multiplex polymerase chain reaction assay was used for diagnosis in 11/12 centers. Eight of 12 centers used inhaled ribavirin (RBV) in some patient populations. Barriers included cost, safety, lack of evidence, and inconvenience. Six of 12 used intravenous immunoglobulin (IVIG), mostly in combination with RBV. Inhaled RBV was used more than oral, and in the post-stem cell transplant population, patients with lower respiratory tract infection (LRTI), graft-versus-host disease, and more recent transplantation were treated at higher rates. Ten centers had experience with lung transplant patients; all used either oral or inhaled RBV for LRTI, 6/10 treated upper respiratory tract infection (URTI). No center treated non-lung solid organ transplant (SOT) recipients with URTI; 7/11 would use oral or inhaled RBV in the same group with LRTI. Patients with hematologic malignancy without hematopoietic stem cell transplantation were treated with RBV at a similar frequency to non-lung SOT recipients. Three of 12 centers, in severe cases, treated parainfluenza and metapneumovirus, and 1/12 treated coronavirus. CONCLUSIONS: Treatment of RSV in immunocompromised patients varied greatly. While most centers treat LRTI, treatment of URTI was variable. No consensus was found regarding the use of oral versus inhaled RBV, or the use of IVIG. The presence of such heterogeneity demonstrates the need for further studies defining optimal treatment of RSV in immunocompromised hosts.

Electrophilic assistance by Asp-99 of 3-oxo-Delta 5-steroid isomerase.

3-Oxo-Delta 5-steroid isomerase (Delta 5-3-ketosteroid isomerase, KSI; EC 5.3.3.1) catalyzes the conversion of a variety of beta, gamma-unsaturated 3-oxosteroids to their corresponding alpha, beta-unsaturated isomers at rates that approach the diffusion limit for specific substrates. The reaction proceeds through a dienolate intermediate, with two amino acid residues (Asp-38 and Tyr-14) known to be involved in catalysis. When the complete three-dimensional structure of KSI was determined recently by NMR methods, an additional polar residue (Asp-99) was found in the active site and this group was shown to be important for catalytic activity. In this work, we examine the properties of several mutant KSIs to determine the nature of catalysis by Asp-99 of KSI. The electrophoretic mobilities of wild-type (WT) KSI and several mutants (D99A, D99N, D38N, and D38N/D99A) on native gels were determined at pH values ranging from 6.0 to 8.5. The results demonstrate that the pKa of Asp-99 is >8.5 in wild-type KSI. The pH-rate profiles for the D99A, D99N, and D38H/D99A mutants of KSI were also determined. For all three mutants, kcat and kcat/KM do not decrease at high pH, in contrast to those for WT and D38H, which lose activity above pH 9 and 8, respectively. Mutation of Asp-99 to Asn decreases kcat for the substrate 5-androstene-3,17-dione by 27-fold and kcat/Km by 23-fold, substantially less than the loss of activity (3000-fold in kcat and 2200-fold in kcat/Km) observed when Asp-99 is mutated to Ala, consistent with a hydrogen bonding role for Asp-99. Taken together, these results provide evidence that Asp-99 participates in catalysis in its protonated form, with a pKa of >9 in WT and approximately 8.5 in the D38H mutant. Asp-99 likely donates a hydrogen bond to O-3 of the steroid, helping to stabilize the transition state(s) of the KSI-catalyzed reaction.

Breathing retraining: a three-year follow-up study of treatment for hyperventilation syndrome and associated functional cardiac symptoms.

This study was designed to evaluate the long-term effects of paced diaphragmatic breathing on subjects who reported functional cardiac symptoms and who also demonstrated associated signs of hyperventilation syndrome. Subjects were a representative sample composed of 10 out of the original 41 subjects who had participated three years previously in a study designed to evaluate the short-term effects of breathing retraining on functional cardiac symptoms and respiratory parameters (respiratory rate and end-tidal carbon dioxide). The results of this follow-up study indicate that breathing retraining had lasting effects on both respiratory parameters measured. Subjects evidenced significantly higher end-tidal carbon dioxide levels and lower respiratory rates when compared to pretreatment levels measured three years earlier. Subjects also continued to report a decrease in the frequency of functional cardiac symptoms when compared to pretreatment levels. We conclude that breathing retraining has lasting effects on respiratory physiology and is highly correlated with a reduction in reported functional cardiac symptoms.

Mechanism of proton transfer in the isomerization of 5-androstene-3, 17-dione by 3-oxo-delta 5-steroid isomerase and its D38E mutant.

The stereochemistry of proton transfer in the isomerization of [4 beta-2H]-5-androstene-3,17- dione (1d) to 4-androstene-3,17-dione (3) catalyzed by 3-oxo-delta 5-steroid isomerase (KSI) has been reinvestigated. In H2O, approximately 65% of the label is retained in the product (3); of this, one-third is at C-4 and two-thirds at C-6 beta. When the same reaction is catalyzed by the D38E mutant of KSI, ca. 60% of the label is retained in the product, but almost all of it is at C-4. These reactions run in deuterium oxide result in 13% incorporation of a second deuterium with the wild type (WT) enzyme and 75% incorporation with the D38E mutant. When unlabeled 1 is isomerized in D2O, there is little incorporation of deuterium with WT (ca. 5 at. %) but substantial incorporation with D38E (130 at. %). These results are consistent with competitive abstraction of both the C-4 alpha and C-4 beta protons, as proposed by Viger et al. [(1981) J. Am. Chem. Soc. 103, 4151], and demonstrate that the KSI reaction is not completely stereospecific. A mechanism is proposed to account for these observations.

Evaluation of the internal equilibrium constant for 3-oxo-delta 5-steroid isomerase using the D38E and D38N mutants: the energetic basis for catalysis.

The dissociation constant (KD) for the complex of the intermediate dienol (2) and the D38N mutant of 3-oxo-delta 5-steroid isomerase (D38N.2) has been determined for the isomerization of 5-androstene-3,17-dione (1). KD for D38N.2 is pH-dependent, with values of 6 nM at pH 6.9, 51 nM at pH 5.8, and 59 nM at pH 5.2. These values of KD are used to estimate the pH-independent dissociation constant (0.7 +/- 0.3 microM) for the complex of dienol and wild-type (WT) enzyme. The internal equilibrium constant (Kint = 0.3 +/- 0.2) for the interconversion of bound substrate (WT.1) and bound intermediate (WT.2) was then calculated for WT using its KD, the values for the external equilibrium constant for 1<-->2, and the dissociation constant of the enzyme substrate complex (KS). The dissociation constant (KD) for the complex of equilenin (4) with WT, D38E, and D38N enzymes was also determined at pH values from 4 to 7. For the complex of 4 with D38N (D38N.4), KD is pH-dependent with an apparent pKa of about 4.5, whereas KD for both WT.4 and D38E.4 is pH-independent. These values are used to give two additional estimates of the internal equilibrium constant for WT (Kint = 0.5 and 0.01). Analysis of these results in terms of Marcus formalism leads to the conclusion that the primary function of the enzyme is to decrease the thermodynamic barrier to formation of the intermediate by lowering delta Gzero by about 10 kcal/mol. In contrast, the intrinsic free energy of activation (delta G++int) is only decreased by about 3 kcal/mol. These results are discussed in terms of competing theories of enzymatic enolization.

Is a proton relay involved in the mechanism of 3-oxo-delta 5-steroid isomerase?

Previous reports of a UV spectral shift upon binding of the competitive inhibitor 19-nortestosterone (1) to 3-oxo-delta 5-steroid isomerase (KSI), coupled with UV resonance Raman results, have led to the conclusion that the enone moiety is polarized to a degree similar to that produced by complete protonation and that a proton relay may be involved in the enzymatic mechanism (Austin et al., 1992). These conclusions were partly based upon interpretations of the corresponding UV spectra of 1 in aqueous acid solutions. These interpretations are shown to be inconsistent with results of deuterium exchange studies and with spectra of model systems. Consequently, there is no evidence either for an extraordinary polarization of 1 produced by binding to the active site of KSI or for a proton relay mechanism.

Energetics of 3-oxo-delta 5-steroid isomerase: source of the catalytic power of the enzyme.

Knowledge of the partitioning of the putative dienol intermediate (2) by steroid isomerase (KSI) (Hawkinson et al. 1991), in conjunction with various steady-state kinetic parameters, allows elucidation of the detailed free energy profile for the KSI-catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3). This free energy profile shows four kinetically significant energy barriers (substrate binding, the two chemical steps, and dissociation of product) that must be traversed upon conversion of 1 to 3. Thus, no single step of the catalytic cycle is cleanly rate-limiting. The source of the catalytic power of KSI is discussed via comparison of the free energy profile for the KSI-catalyzed isomerization with those for the acetate-catalyzed isomerization and the aqueous reaction at pH 7. Similarities between the energetics of the KSI-catalyzed and triosephosphate isomerase catalyzed reactions are also noted.

Kinetic competence of an externally generated dienol intermediate with steroid isomerase.

The putative intermediate dienol (2) in the steroid isomerase (KSI) catalyzed conversion of 5-androstene-3,17-dione (1) to 4-androstene-3,17-dione (3) has been independently generated and tested as a substrate for KSI. At pH 7, dienol 2 is converted by KSI to a mixture of 1 (46%) and 3 (54%). The apparent second-order rate constant for reaction of 2 with KSI to produce 3 (kappa cat/Km = 2.3 x 10(8) M-1 s-1) is similar to that for reaction of 1 with KSI (kappa cat/Km = 2.1 x 10(8) M-1 s-1), demonstrating that 2 is kinetically competent. Isomerization of 1 by KSI in D2O gives only 5% of solvent deuterium incorporated into the product 3. When 2 reacts with KSI in D2O, and the product 3 is isolated (from direct reaction of 2 and from subsequent conversion of the 1 initially formed), ca. 80 atom % deuterium is located at C-6 beta, confirming that protonation of the dienol by KSI occurs at the same face as the proton transfer in the KSI catalyzed reaction of 1 to 3.