False-positive hepatitis C virus serology after placement of a ventricular assistance device.

BACKGROUND: Ventricular assist devices (VADs) have been associated with immune activation and sensitization. We observed several cases of false-positive (FP) hepatitis C virus (HCV) antibody (Ab) tests in patients being evaluated for orthotopic heart transplant (OHT), prompting us to investigate this further. METHODS: We reviewed all VAD and OHT cases at Johns Hopkins from 2005 to 2012. FP HCV serology was defined as an equivocal or low-positive HCV Ab, plus either (i) a negative recombinant immunoblot (RIBA) and/or HCV nucleic acid test (NAT), or (ii) an indeterminate RIBA and negative NAT. RESULTS: In 53 patients with available HCV testing, nearly 40% of patients (21/53: 39.6%) developed FP HCV Ab tests after VAD placement: 4 patients had negative NAT, 12 had negative RIBA, and 5 had an indeterminate RIBA and negative NAT. All patients with indeterminate RIBA tests had isolated reactivity to the same HCV protein, c100p/5-1-1p (NS4b protein). In 3 of 4 VAD patients who had OHT and repeat HCV Ab testing after VAD removal, repeat HCV Ab was negative (699-947 days after OHT); in 1 case, FP HCV serology persisted (5 days after OHT). Thirteen patients had OHT alone and none developed a FP HCV Ab. CONCLUSIONS: FP HCV Ab results following VAD placement are very common. Reversal of FP serology in several patients after VAD removal is suggestive of a possible association with the VAD hardware. Clinicians should be aware of this phenomenon, as it could lead to delays in determining eligibility for OHT and increased costs.

Posaconazole liquid suspension in solid organ transplant recipients previously treated with voriconazole.

BACKGROUND: Posaconazole (PCZ) has become an attractive alternative to voriconazole (VCZ) in transplant recipients with suspected or proven invasive filamentous fungal infections, causing fewer drug interactions. Here, we describe our experience with PCZ after VCZ in solid organ transplant (SOT) recipients. METHODS: VCZ was replaced by PCZ liquid solution in 19 SOT recipients (15 lung, 2 kidney, 1 liver, and 1 heart/lung) with invasive pulmonary aspergillosis (12/19; 63.2%), possible invasive pulmonary fungal infection (2/19; 10.5%), prophylaxis (2/19; 10.5%), or pulmonary scedosporiosis, mucormycosis, and mixed fungal species (1 each). Rationales for switch were suspected adverse reactions to VCZ (17/19; 89.4%) and desire to broaden spectrum of coverage to include agents of mucormycosis (3/19; 15.8%). RESULTS: PCZ was well tolerated in all patients. In those patients with baseline liver enzyme abnormalities, a median change occurred in concentrations of alanine transaminase (-20 IU/L), aspartate aminotransferase (-17.5 IU/L), and alkaline phosphatase (-61.5 IU/L). Clinical success (resolution, stabilization, or prevention of infection) was achieved in 16/19 (84%) people. CONCLUSION: PCZ appears to have a reasonable safety and tolerability profile and may be an effective alternative in SOT patients who require an agent with anti-mold activity, but are unable to tolerate VCZ.

Voriconazole therapeutic drug monitoring: results of a prematurely discontinued randomized multicenter trial.

BACKGROUND: Voriconazole (VOR) levels are highly variable, with potential implications to both efficacy and safety. We hypothesized that VOR therapeutic drug monitoring (TDM) will decrease the incidence of treatment failures and adverse events (AEs). METHODS: We initiated a prospective, randomized, non-blinded multicenter study to compare clinical outcomes in adult patients randomized to standard dosing (clinician-driven) vs. TDM (doses adjusted based on levels). VOR trough levels were obtained on day 5, 14, 28, and 42 (or at completion of drug; ± 3 days). Real-time dose adjustments were made to maintain a range between 1-5 µg/mL on the TDM-arm, while levels were assessed retrospectively in the standard-arm. Patient questionnaires were administered to assess subjective AEs. RESULTS: The study was discontinued prematurely, after 29 patients were enrolled. Seventeen (58.6%) patients experienced 38 AEs: visual changes (22/38, 57.9%), neurological symptoms (13/38, 34.2%), and liver abnormalities (3/38, 7.9%). VOR was discontinued in 7 (25%) patients because of an AE (4 standard-arm, 3 TDM-arm). VOR levels were frequently out of range in the standard-arm (8 tests >5 µg/mL; 9 tests <1 µg/mL). Three dose changes occurred in the TDM-arm for VOR levels <1 µg/mL. Levels decreased over time in the standard-arm, with mean VOR levels lower at end of therapy compared to TDM (1.3 vs. 4.6 µg/mL, P = 0.008). CONCLUSIONS: VOR TDM has become widespread clinical practice, based on known variability in drug levels, which impaired accrual in this study. Although comparative conclusions are limited, observations of variability and waning levels over time support TDM.

Epidemiology, outcomes, and mortality predictors of invasive mold infections among transplant recipients: a 10-year, single-center experience.

BACKGROUND: The epidemiology of invasive mold infections (IMI) in transplant recipients differs based on geography, hosts, preventative strategies, and methods of diagnosis. METHODS: We conducted a retrospective observational study to evaluate the epidemiology of proven and probable IMI, using prior definitions, among all adult hematopoietic stem cell transplant (HSCT) and solid organ transplant (SOT) recipients in the era of "classic" culture-based diagnostics (2000-2009). Epidemiology was evaluated before and after an initiative was begun to increase bronchoscopy in HSCT recipients after 2005. RESULTS: In total, 106 patients with one IMI were identified. Invasive aspergillosis (IA) was the most common IMI (69; 65.1%), followed by mucormycosis (9; 8.5%). The overall rate of IMI (and IA) was 3.5% (2.5%) in allogeneic HSCT recipients. The overall incidence for IMI among lung, kidney, liver, and heart transplant recipients was 49, 2, 11, and 10 per 1000 person-years, respectively. The observed rate of IMI among human leukocyte antigen-matched unrelated and haploidentical HSCT recipients increased from 0.6% annually to 3.0% after bronchoscopy initiation (P < 0.05). The 12-week mortality among allogeneic HSCT, liver, kidney, heart, and lung recipients with IMI was 52.4%, 47.1%, 27.8%, 16.7%, and 9.5%, respectively. Among allogeneic HSCT (odds ratio [OR]: 0.07, P = 0.007) and SOT (OR: 0.22, P = 0.05) recipients with IA, normal platelet count was associated with improved survival. Male gender (OR: 14.4, P = 0.007) and elevated bilirubin (OR: 5.7, P = 0.04) were significant predictors of mortality for allogeneic HSCT and SOT recipients with IA, respectively. CONCLUSIONS: During the era of culture-based diagnostics, observed rates of IMI were low among all transplants except lung transplant recipients, with relatively higher mortality rates. Diagnostic aggressiveness and host variables impact the reported incidence and outcome of IMI and likely account for institutional variability in multicenter studies. Definitions to standardize diagnoses among SOT recipients are needed.

Epidemiology, risk factors, and outcomes of Clostridium difficile infection in kidney transplant recipients.

BACKGROUND: We sought to describe the epidemiology and risk factors for Clostridium difficile infection (CDI) among kidney transplant recipients (KTR) between 1 January 2008 and 31 December 2010. METHODS: A single-institution retrospective study was conducted among all adult KTR with CDI, defined as a positive test for C. difficile by a cell cytotoxic assay for C. difficile toxin A or B or polymerase chain reaction test for toxigenic C. difficile. RESULTS: Among 603 kidney transplants performed between 1 January 2008 and 31 December 2010, 37 (6.1%) patients developed CDI: 12 (of 128; 9.4%) high-risk (blood group incompatible and/or anti-human leukocyte antigen donor-specific antibodies) vs. 25 (of 475; 5.3%, P = 0.08) standard-risk patients. The overall rate of CDI increased from 3.7% in 2008 to 9.4% in 2010 (P = 0.05). The median time to CDI diagnosis was 9 days, with 27 (73.0%) patients developing CDI within the first 30 days after their transplant, and 14 (51.8%) developing CDI within 7 days. A case-control analysis of 37 CDI cases and 74 matched controls demonstrated the following predictors for CDI among KTR: vancomycin-resistant Enterococcus colonization before transplant (odds ratio [OR]: 3.6, P = 0.03), receipt of an organ from Centers for Disease Control high-risk donor (OR: 5.9, P = 0.006), and administration of high-risk antibiotics within 30 days post transplant (OR: 6.6, P = 0.001). CONCLUSIONS: CDI remains a common early complication in KTR, with rates steadily increasing during the study period. Host and transplant-related factors and exposure to antibiotics appeared to significantly impact the risk for CDI among KTR.

Characterization of the Candida albicans TRP1 gene and construction of a homozygous trp1 mutant by sequential co-transformation.

The Candida albicans TRP1 gene has been isolated by complementation of an Escherichia coli trpC mutant. Sequence analysis has revealed a single ORF (open reading frame) of 678 nucleotides (nt). The amino acid (aa) sequence deduced from this coding region demonstrates a high degree of homology with PRAI (phosphoribosylanthranilate isomerase) enzymes of other fungi, as well as bacterial species. The gene is also analogous to other yeast TRP1 genes in that it encodes a unifunctional enzyme, whereas TRP1 in filamentous fungi encodes a tri-functional enzyme. Both chromosomal copies of the gene were disrupted by sequential integrative transformation employing co-transformation of an ade1 mutant in order to create a homozygous auxotrophic trp1,ade1 C. albicans strain. This double auxotroph was used to test the ability of the Saccharomyces cerevisiae TRP1 gene to complement the C. albicans trp1 mutation; no expression of the S. cerevisiae gene was detectable.

A rapid method for spin-drying large quantities of cytofunnels.

A centrifugal spinner designed for drying lettuce leaves has been found to facilitate rapid drying of large quantities of cytofunnels.

The Stressors of Clergy Children Inventory: reliability and validity.

The purpose of this study was to develop and evaluate the Stressors of Clergy Children Inventory. The initial self-report survey was tested for internal consistency reliability. Tests for construct validity, concurrent validity, and internal consistency reliability indicated the inventory could be used in research. Recommendations for refinement and use were presented.

Reliability and validity of the Adolescent Family Life Satisfaction Index.

The purpose of this study was to develop and evaluate the Adolescent Family Life Satisfaction Index. The self-report questionnaire was tested for internal consistency reliability. Tests for construct validity, concurrent validity, and internal consistency reliability provided support for the use of the over-all Adolescent Family Life Satisfaction Index, Parental Subscale, and Sibling Subscale for the measurement of adolescents' reports of satisfaction with family life.

Isolation and characterization of the Candida albicans PFY1 gene for profilin.

We have isolated the Candida albicans gene for profilin, PFY1. Degenerate oligonucleotide primers based on regions of high homology were utilized to obtain a polymerase chain reaction-amplified copy of the gene. This was then used as a probe to isolate the gene from a C. albicans genomic library. Our studies indicate that the full-length gene is unstable in Escherichia coli. Several clones were sequenced, and the predicted amino acid sequence demonstrated homology with profilin proteins from other organisms, most notably Saccharomyces cerevisiae. Northern analysis revealed that the gene is expressed in C. albicans. Attempts to express the gene in S. cerevisiae cells were unsuccessful until the C. albicans promoter was replaced with an S. cerevisiae promoter. Functional complementation of the gene was demonstrated in S. cerevisiae profilin-requiring cells. Antibodies raised to isolated C. albicans profilin protein recognized a protein of the predicted molecular weight when the gene was expressed in S. cerevisiae cells.

The extracellular domain of the Saccharomyces cerevisiae Sln1p membrane osmolarity sensor is necessary for kinase activity.

The function of the extracellular domain (ECD) of Sln1p, a plasma membrane two-transmembrane domain (TMD) sensor of the high-osmolarity glycerol (HOG) response pathway, has been studied in the yeast Saccharomyces cerevisiae. Truncations of SLN1 that retain an intact kinase domain are capable of complementing the lethality of an sln1Delta strain. By observing levels of Hog1p phosphorylation as well as the phosphorylation state of Sln1p, the kinase activities of various SLN1 constructions were determined. In derivatives that do not contain the first TMD, Sln1p activity was no longer dependent on medium osmolarity but appeared to be constitutively active even under conditions of high osmolarity. Removal of the first TMD (DeltaTMD1 construct) gave a protein that was strongly phosphorylated whereas Hog1p was largely dephosphorylated, as expected if the active form of Sln1p is phosphorylated. When both TMDs as well as the ECD were deleted, so that the kinase domain is cytosolic, Sln1p was not phosphorylated whereas Hog1p became constitutively hyperphosphorylated. Surprisingly, this hyperactivity of the HOG mitogen-activated protein kinase signaling pathway was not sufficient to result in cell lethality. When the ECD of the DeltaTMD1 construct was replaced with a leucine zipper motif, Sln1p was hyperactive, so that Hog1p became mostly unphosphorylated. In contrast, when the Sln1p/leucine zipper construct was crippled by a mutation of one of the internal leucines, the Sln1 kinase was inactive. These experiments are consistent with the hypothesis that the ECD of Sln1p functions as a dimerization and activation domain but that osmotic regulation of activity requires the presence of the first TMD.

Regulation of profilin localization in Saccharomyces cerevisiae by phosphoinositide metabolism.

Profilin is an actin- and phosphatidylinositol 4,5-bisphosphate-binding protein that plays a role in the organization of the cytoskeleton and may be involved in growth factor signaling pathways. The subcellular localization of profilin was examined in the yeast Saccharomyces cerevisiae. Immunoblot analysis showed that profilin was localized in both the plasma membrane and cytosolic fractions of the cell. Actin was bound to the profilin localized in the cytosol. The association of profilin with the membrane was peripheral and mediated through interaction with phospholipid. The phospholipid dependence of profilin for membrane binding was examined in vitro using pure profilin and defined unilamellar phospholipid vesicles. The presence of phosphatidylinositol 4,5-bisphosphate in phospholipid vesicles was required for maximum profilin binding. Moreover, the binding of profilin to phospholipid vesicles was dependent on the surface concentration of phosphatidylinositol 4,5-bisphosphate. The subcellular localization of profilin was examined in vivo under growth conditions (i.e. inositol starvation of ino1 cells and glucose starvation of respiratory deficient cells) where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were depleted. Depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate levels resulted in a translocation of profilin from the plasma membrane to the cytosolic fraction. Profilin translocated back to the membrane fraction from the cytosol under growth conditions where plasma membrane levels of phosphatidylinositol 4,5-bisphosphate were replenished. These results suggested that phosphoinositide metabolism played a role in the localization of profilin.

Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis.

Apoptosis has been identified recently as a component of many cardiac pathologies. However, the potential triggers of programmed cell death in the heart and the involvement of specific metabolic pathway(s) are less well characterized. Detachment of cytochrome c from the mitochondrial inner membrane is a necessary first step for cytochrome c release into the cytosol and initiation of apoptosis. The saturated long chain fatty acid, palmitate, induces apoptosis in rat neonatal cardiomyocytes and diminishes content of the mitochondrial anionic phospholipid, cardiolipin. These changes are accompanied by 1) acyl chain saturation of phosphatidic acid and phosphatidylglycerol, 2) large increases in the levels of these two phospholipids, and 3) a decline in cardiolipin synthesis. Although cardiolipin synthase activity is unchanged, saturated phosphatidylglycerol is a poor substrate for this enzyme. Under these conditions, decreased cardiolipin synthesis and release of cytochrome c are directly and significantly correlated. The results suggest that phosphatidylglycerol saturation and subsequent decreases in cardiolipin affect the association of cytochrome c with the inner mitochondrial membrane, directly influencing the pathway to cytochrome c release and subsequent apoptosis.

Lack of mitochondrial anionic phospholipids causes an inhibition of translation of protein components of the electron transport chain. A yeast genetic model system for the study of anionic phospholipid function in mitochondria.

Reduction of mitochondrial cardiolipin (CL) levels has been postulated to compromise directly the function of several essential enzymes and processes of the mitochondria. There is limited genetic evidence for the critical roles with which CL and its precursor phosphatidylglycerol (PG) have been associated. A null allele of the PGS1 gene from Saccharomyces cerevisiae, which encodes the enzyme responsible for the synthesis of the CL precursor PG phosphate, was created in a yeast strain in which PGS1 expression is exogenously regulated by doxycycline. The addition of increasing concentrations of doxycycline to the growth medium causes a proportional decrease to undetectable levels of PGS1 transcript, PG phosphate synthase activity, and PG plus CL. The doubling time of this strain with increasing doxycycline increases to senescence in non-fermentable carbon sources or at high temperatures, conditions that do not support growth of the pgs1Delta strain. Doxycycline addition also causes mitochondrial abnormalities as observed by fluorescence microscopy. Products of four mitochondrial encoded genes (COX1, COX2, COX3, and COB) and one nuclear encoded gene (COX4) associated with the mitochondrial inner membrane are not present when PGS1 expression is fully repressed. No translation of these proteins can be detected in cells lacking the PGS1 gene product, although transcription and splicing appear unaffected. Protein import of other nuclear encoded proteins remains unaffected. The remaining proteins encoded by mitochondrial DNA are expressed and translated normally. Thus, the molecular basis for the lack of mitochondrial function in pgs1Delta cells is the failure to translate gene products essential to the electron transport chain.

Polyproline binding is an essential function of human profilin in yeast.

Structural analysis of human profilin has revealed two tryptophan residues, W3 and W31, which interact with polyproline. The codons for these residues were mutated to encode phenylalanine and the mutant proteins overexpressed in Eschericia coli. The isolated proteins were diminished in their ability to bind polyproline, whereas phosphatidylinositol 4,5-bisphosphate (PIP2) binding remained unchanged. In many strains of Saccharomyces cerevisiae, disruption of the gene encoding profilin, PFY1, is lethal. It was found that expression of the gene for human profilin is capable of suppressing this lethality. The polyproline-binding mutant alleles of the human gene were cloned into various yeast expression vectors. Each of the mutant genes resulted in suppression of the lethality of pfy1Delta. It was observed that the mutant protein expression levels paralleled the growth rates of the strains. The severity of various morphological abnormalities of the strains was also attenuated with increased protein levels, suggesting that profilin polyproline-binding mutations are deleterious to cell growth unless overexpressed. Both tryptophan mutations were combined to give a third mutant allele that was found both unable to bind polyproline and to suppress the lethality of a pfy1 deletion. Immunoprecipitation experiments suggested that the mutants were unaltered in their affinity for actin and PIP2. These data strongly suggest that polyproline binding is an essential function of profilin.

Effect of CTP synthetase regulation by CTP on phospholipid synthesis in Saccharomyces cerevisiae.

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) activity in Saccharomyces cerevisiae is allosterically regulated by CTP product inhibition. Amino acid residue Glu161 in the URA7-encoded and URA8-encoded CTP synthetases was identified as being involved in the regulation of these enzymes by CTP product inhibition. The specific activities of the URA7-encoded and URA8-encoded enzymes with a Glu161 --> Lys (E161K) mutation were 2-fold greater when compared with the wild-type enzymes. The E161K mutant URA7-encoded and URA8-encoded CTP synthetases were less sensitive to CTP product inhibition with inhibitor constants for CTP of 8.4- and 5-fold greater, respectively, than those of their wild-type counterparts. Cells expressing the E161K mutant enzymes on a multicopy plasmid exhibited an increase in resistance to the pyrimidine poison and cancer therapeutic drug cyclopentenylcytosine and accumulated elevated (6-15-fold) levels of CTP when compared with cells expressing the wild-type enzymes. Cells expressing the E161K mutation in the URA7-encoded CTP synthetase exhibited an increase (1.5-fold) in the utilization of the Kennedy pathway for phosphatidylcholine synthesis when compared with control cells. Cells bearing the mutation also exhibited an increase in the synthesis of phosphatidylcholine (1.5-fold), phosphatidylethanolamine (1.3-fold), and phosphatidate (2-fold) and a decrease in the synthesis of phosphatidylserine (1.7-fold). These alterations were accompanied by an inositol excretion phenotype due to the misregulation of the INO1 gene. Moreover, cells bearing the E161K mutation exhibited an increase (1.6-fold) in the ratio of total neutral lipids to phospholipids, an increase in triacylglycerol (1.4-fold), free fatty acids (1.7-fold), and ergosterol ester (1.8-fold), and a decrease in diacylglycerol (1. 3-fold) when compared with control cells. These data indicated that the regulation of CTP synthetase activity by CTP plays an important role in the regulation of phospholipid synthesis.

Intravenous vs. left ventricular injection of ionic contrast material: hemodynamic implications for digital subtraction angiography.

Because of the increased use of intravenous injection of contrast material for the evaluation of cardiac structure and function by digital subtraction techniques, a study was done to assess the hemodynamic effects of contrast material when used in this fashion in man. In 10 patients, with each serving as his own control, the effects of intravenous and intraventricular injections of sodium meglumine diatrizoate (Renografin 76) in the same dose were compared. There was no difference between these two methods with respect to changes in pulmonary wedge pressures, systemic pressures, and pulmonary vascular resistance. The elevation of mean pulmonary artery and right atrial pressure was greater after the intraventricular injection (p less than 0.05). The elevated cardiac output and systemic vascular resistance returned to control values somewhat more quickly after the intravenous injection (p less than 0.001 and p less than 0.05, respectively); and the increase in cardiac output was greater after the intravenous injection at 1 min (p less than 0.05), but less than after the intraventricular injection at 2 min (p less than 0.05). Despite the detection of these statistically significant differences, the magnitude and timing of these differences are too small to justify the notion that imaging by intravenous injections of standard ionic contrast media provides any substantial hemodynamic benefits or decreased risk to the patient.

Identification of Ser424 as the protein kinase A phosphorylation site in CTP synthetase from Saccharomyces cerevisiae.

The URA7-encoded CTP synthetase [EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)] in the yeast Saccharomyces cerevisiae is phosphorylated on a serine residue and stimulated by cAMP-dependent protein kinase (protein kinase A) in vitro. In vivo, the phosphorylation of CTP synthetase is mediated by the RAS/cAMP pathway. In this work, we examined the hypothesis that amino acid residue Ser424 contained in a protein kinase A sequence motif in the URA7-encoded CTP synthetase is the target site for protein kinase A. A CTP synthetase synthetic peptide (SLGRKDSHSA) containing the protein kinase A motif was a substrate (Km = 30 microM) for protein kinase A. This peptide also inhibited (IC50 = 45 microM) the phosphorylation of purified wild-type CTP synthetase by protein kinase A. CTP synthetase with a Ser424 --> Ala (S424A) mutation was constructed by site-directed mutagenesis. The mutated enzyme was not phosphorylated in response to the activation of protein kinase A activity in vivo. Purified S424A mutant CTP synthetase was not phosphorylated and stimulated by protein kinase A. The S424A mutant CTP synthetase had reduced Vmax and elevated Km values for ATP and UTP when compared with the protein kinase A-phosphorylated wild-type enzyme. The specificity constants for ATP and UTP for the S424A mutant CTP synthetase were 4.2- and 2.9-fold lower, respectively, when compared with that of the phosphorylated enzyme. In addition, the S424A mutant enzyme was 2.7-fold more sensitive to CTP product inhibition when compared with the phosphorylated wild-type enzyme. These data indicated that the protein kinase A target site in CTP synthetase was Ser424 and that the phosphorylation of this site played a role in the regulation of CTP synthetase activity.