Spectral features of non-nutritive suck dynamics in extremely preterm infants.

Background: Non-nutritive suck (NNS) is used to promote ororhythmic patterning and assess oral feeding readiness in preterm infants in the neonatal intensive care unit (NICU). While time domain measures of NNS are available in real time at cribside, our understanding of suck pattern generation in the frequency domain is limited. The aim of this study is to model the development of NNS in the frequency domain using Fourier and machine learning (ML) techniques in extremely preterm infants (EPIs). Methods: A total of 117 EPIs were randomized to a pulsed or sham orocutaneous intervention during tube feedings 3 times/day for 4 weeks, beginning at 30 weeks post-menstrual age (PMA). Infants were assessed 3 times/week for NNS dynamics until they attained 100% oral feeding or NICU discharge. Digitized NNS signals were processed in the frequency domain using two transforms, including the Welch power spectral density (PSD) method, and the Yule-Walker PSD method. Data analysis proceeded in two stages. Stage 1: ML longitudinal cluster analysis was conducted to identify groups (classes) of infants, each showing a unique pattern of change in Welch and Yule-Walker calculations during the interventions. Stage 2: linear mixed modeling (LMM) was performed for the Welch and Yule-Walker dependent variables to examine the effects of gestationally-aged (GA), PMA, sex (male, female), patient type [respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD)], treatment (NTrainer, Sham), intervention phase [1, 2, 3], cluster class, and phase-by-class interaction. Results: ML of Welch PSD method and Yule-Walker PSD method measures revealed three membership classes of NNS growth patterns. The dependent measures peak_Hz, PSD amplitude, and area under the curve (AUC) are highly dependent on PMA, but show little relation to respiratory status (RDS, BPD) or somatosensory intervention. Thus, neural regulation of NNS in the frequency domain is significantly different for each identified cluster (classes A, B, C) during this developmental period. Conclusions: Efforts to increase our knowledge of the evolution of the suck central pattern generator (sCPG) in preterm infants, including NNS rhythmogenesis will help us better understand the observed phenotypes of NNS production in both the frequency and time domains. Knowledge of those features of the NNS which are relatively invariant vs. other features which are modifiable by experience will likewise inform more effective treatment strategies in this fragile population.

Assessment of microplastics in Irish river sediment.

Microplastics (MPs) are environmental pollutants of growing concern, and awareness of MPs pollution in marine and freshwater environments has increased in recent years. However, knowledge of MPs contamination in riverine sediments in Ireland is limited. To address this, we collected and analysed sediment samples from 16 selected sites along the River Barrow. Microplastics were extracted through a density separation method, after which their size, colour, and shape were analysed under a stereo microscope (Optica SZM-2). Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to identify polymer types. A total of 690 MPs were recovered from the 16 sites, with fibres as the dominant MP type. The highest concentration of MPs was 155 MP fibres kg-1 wet sediment found in samples collected from Graiguenamanagh, Co. Kilkenny (GK). The majority of the recovered MPs were polyethylene (PE), polypropylene (PP), nylon, and cellulose acetate (CA) fibres. Overall, this study highlighted the presence of MPs in Irish river sediments and provided a baseline for future studies on MPs pollution. Further research is needed to better understand sources, distribution, and effects of MPs in freshwater ecosystems.

Cardiac Interventions for Patients With Trisomy 13 and Trisomy 18: Experience, Ethical Issues, Communication, and the Case for Individualized Family-Centered Care.

This report is informed by the themes of the session Trisomy 13/18, Exploring the Changing Landscape of Interventions at NeoHeart 2020-The Fifth International Conference of the Neonatal Heart Society. The faculty reviewed the present evidence in the management of patients and the support of families in the setting of trisomy 13 and trisomy 18 with congenital heart disease. Until recently medical professionals were taught that T13 and 18 were "lethal conditions" that were "incompatible with life" for which measures to prolong life are therefore ethically questionable and likely futile. While the medical literature painted one picture, family support groups shared stories of the long-term survival of children who displayed happiness and brought joy along with challenges to families. Data generated from such care shows that surgery can, in some cases, prolong survival and increase the likelihood of time at home. The authors caution against a change from never performing heart surgery to always-we suggest that the pendulum of intervention find a balanced position where all therapies including comfort care and surgery can be reviewed. Families and clinicians should typically be supported and empowered to define the best care for their children and patients. Key concepts in communication and case vignettes are reviewed including the importance of supportive relationships and the fact that palliative care may serve as an additional layer of support for decision-making and quality of life interventions. While cardiac surgery may be beneficial in some cases, surgery should not be the primary focus of initial family education and support.

Effect of Whole-Genome Sequencing on the Clinical Management of Acutely Ill Infants With Suspected Genetic Disease: A Randomized Clinical Trial.

Importance: Whole-genome sequencing (WGS) shows promise as a first-line genetic test for acutely ill infants, but widespread adoption and implementation requires evidence of an effect on clinical management. Objective: To determine the effect of WGS on clinical management in a racially and ethnically diverse and geographically distributed population of acutely ill infants in the US. Design, Setting, and Participants: This randomized, time-delayed clinical trial enrolled participants from September 11, 2017, to April 30, 2019, with an observation period extending to July 2, 2019. The study was conducted at 5 US academic medical centers and affiliated children's hospitals. Participants included infants aged between 0 and 120 days who were admitted to an intensive care unit with a suspected genetic disease. Data were analyzed from January 14 to August 20, 2020. Interventions: Patients were randomized to receive clinical WGS results 15 days (early) or 60 days (delayed) after enrollment, with the observation period extending to 90 days. Usual care was continued throughout the study. Main Outcomes and Measures: The main outcome was the difference in the proportion of infants in the early and delayed groups who received a change of management (COM) 60 days after enrollment. Additional outcome measures included WGS diagnostic efficacy, within-group COM at 90 days, length of hospital stay, and mortality. Results: A total of 354 infants were randomized to the early (n = 176) or delayed (n = 178) arms. The mean participant age was 15 days (IQR, 7-32 days); 201 participants (56.8%) were boys; 19 (5.4%) were Asian; 47 (13.3%) were Black; 250 (70.6%) were White; and 38 (10.7%) were of other race. At 60 days, twice as many infants in the early group vs the delayed group received a COM (34 of 161 [21.1%; 95% CI, 15.1%-28.2%] vs 17 of 165 [10.3%; 95% CI, 6.1%-16.0%]; P = .009; odds ratio, 2.3; 95% CI, 1.22-4.32) and a molecular diagnosis (55 of 176 [31.0%; 95% CI, 24.5%-38.7%] vs 27 of 178 [15.0%; 95% CI, 10.2%-21.3%]; P < .001). At 90 days, the delayed group showed a doubling of COM (to 45 of 161 [28.0%; 95% CI, 21.2%-35.6%]) and diagnostic efficacy (to 56 of 178 [31.0%; 95% CI, 24.7%-38.8%]). The most frequent COMs across the observation window were subspecialty referrals (39 of 354; 11%), surgery or other invasive procedures (17 of 354; 4%), condition-specific medications (9 of 354; 2%), or other supportive alterations in medication (12 of 354; 3%). No differences in length of stay or survival were observed. Conclusions and Relevance: In this randomized clinical trial, for acutely ill infants in an intensive care unit, introduction of WGS was associated with a significant increase in focused clinical management compared with usual care. Access to first-line WGS may reduce health care disparities by enabling diagnostic equity. These data support WGS adoption and implementation in this population. Trail Registration: ClinicalTrials.gov Identifier: NCT03290469.

Management preferences in ECMO mode for congenital diaphragmatic hernia.

PURPOSE: The purpose of this study was to identify management preferences that may exist in the care of infants with CDH receiving ECMO with emphasis on VV-ECMO. METHODS: A survey was created to measure treatment preferences regarding ECMO use in CDH. The survey was distributed to all APSA and ELSO/Euro-ELSO members via e-mail. Survey results were summarized using descriptive statistics. RESULTS: The survey had 230 respondents. The survey participants were surgeons (75%), neonatologists/intensivists (23%), and "other" (2%). The mean annual center volume was 11.6(±9.6) CDH cases, and the average number treated with ECMO was 4.5 (±6.4) cases/yr. The most agreed upon criteria for ECMO initiation were preductal O2 saturation <80% refractory to ventilator manipulation and medical therapy (89%), oxygenation index >40 (80%), severe air-leak (79%), and mixed acidosis (75%). Over 60% of respondents agreed the VV-ECMO would be optimum for average risk neonates. However, this preference diminished as the pre-ECMO level of cardiac support increased. When asked about why each respondent would choose VA-ECMO over VV-ECMO, the responses varied significantly between surgeons and non-surgeons. CONCLUSION: While there seem to be areas of consensus among practitioners, such as criteria for initiation of ECMO, this survey revealed substantial variation in individual practice patterns regarding the use of ECMO for CDH. TYPE OF STUDY: Qualitative, Survey. LEVEL OF EVIDENCE: IV.

Outcomes of infants with congenital diaphragmatic hernia treated with venovenous versus venoarterial extracorporeal membrane oxygenation: A propensity score approach.

PURPOSE: Previous studies comparing extracorporeal membrane oxygenation (ECMO) modality for congenital diaphragmatic hernia (CDH) have not accounted for confounding by indication. We therefore hypothesized that using a propensity score (PS) approach to account for selection bias may identify outcome differences based on ECMO modality for infants with CDH. METHODS: We utilized ELSO Registry data (2000-2016). Patients with CDH were divided to either venoarterial (VA) or venovenous (VV) ECMO. Patients were matched by PS to control for nonrandom treatment assignment. Subgroup analyses were conducted based on timing of CDH repair relative to ECMO. Primary analysis was the "intent-to-treat" cohort based on the initial ECMO mode. Mortality was the primary outcome, and severe neurologic injury (SNI) was a secondary outcome. RESULTS: PS matching (3:1) identified 3304 infants (VA = 2470, VV = 834). In the main group, mortality was not different between VA and VV ECMO (OR = 1.01, 95% CI: 0.86-1.18) and there was no difference in SNI between VA and VV (OR = 0.80; 95% CI: 0.63-1.01). For the pre-ECMO CDH repair subgroup, 175 VA cases were matched to 70 VV. In these neonates, mortality was higher for VV compared to VA (OR = 2.10, 95% CI: 1.19-3.69), without any difference in SNI (OR = 1.48; 95% CI: 0.59-3.71). For the subgroup that did not have pre-ECMO CDH repair, 2030 VA cases were matched to 683 VV cases. In this subgroup, VV was associated with 27% lower risk of SNI relative to VA (OR = 0.73, 95% CI: 0.56-0.95) without any difference in mortality (OR = 0.94, 95% CI: 0.79-1.11). CONCLUSION: This study revalidates that ECMO mode does not significantly affect mortality or SNI in infants with CDH. In the subset of infants who require pre-ECMO CDH repair, VA favors survival, whereas, in the subgroup of infants that did not have pre-ECMO CDH repair, VV favors lower rates of SNI. We conclude that neither mode appears consistently superior across all situations, and clinical judgment should remain a multifactorial decision. LEVEL OF EVIDENCE: Level III.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9.

Transcription of expanded microsatellite repeats is associated with multiple human diseases, including myotonic dystrophy, Fuchs endothelial corneal dystrophy, and C9orf72-ALS/FTD. Reducing production of RNA and proteins arising from these expanded loci holds therapeutic benefit. Here, we tested the hypothesis that deactivated Cas9 enzyme impedes transcription across expanded microsatellites. We observed a repeat length-, PAM-, and strand-dependent reduction of repeat-containing RNAs upon targeting dCas9 directly to repeat sequences; targeting the non-template strand was more effective. Aberrant splicing patterns were rescued in DM1 cells, and production of RAN peptides characteristic of DM1, DM2, and C9orf72-ALS/FTD cells was drastically decreased. Systemic delivery of dCas9/gRNA by adeno-associated virus led to reductions in pathological RNA foci, rescue of chloride channel 1 protein expression, and decreased myotonia. These observations suggest that transcription of microsatellite repeat-containing RNAs is more sensitive to perturbation than transcription of other RNAs, indicating potentially viable strategies for therapeutic intervention.

Cannulating the contraindicated: effect of low birth weight on mortality in neonates with congenital diaphragmatic hernia on extracorporeal membrane oxygenation.

BACKGROUND/PURPOSE: Restrictions for ECMO in neonates include birth weight less than 2kg (BW <2kg) and/or gestational age less than 34weeks (GA <34weeks). We sought to describe their relationship on mortality. METHODS: Neonates with a primary diagnosis code of CDH were identified in the Extracorporeal Life Support Organization (ELSO) registry, and logistic regression models were used to examine the effect of BW <2kg and GA <34weeks on mortality. RESULTS: We identified 7564 neonates with CDH. The overall mortality was 50%. There was a significantly higher risk of death with unadjusted odds ratio (OR) 2.39 (95% confidence interval [CI]: 1.53-3.74; P<0.01) for BW <2kg neonates. The adjusted OR of death for BW <2kg neonates remained significantly high with over two-fold increase in the odds of mortality when adjusted for potential confounding variables (OR 2.11, 95% CI: 1.30-3.43; P<0.01). However, no difference in mortality was observed in neonates with GA <34weeks. CONCLUSIONS: While mortality among CDH neonates with a BW <2kg was substantially increased, GA <34weeks was not significantly associated with mortality. Effort should be made to identify the best candidates for ECMO in this high-risk group and develop treatment strategies to optimize their survival. TYPE OF STUDY: Case-Control Study, Retrospective Comparative Study. LEVEL OF EVIDENCE: Level III.

Tumor-suppressor genes that escape from X-inactivation contribute to cancer sex bias.

There is a striking and unexplained male predominance across many cancer types. A subset of X-chromosome genes can escape X-inactivation, which would protect females from complete functional loss by a single mutation. To identify putative 'escape from X-inactivation tumor-suppressor' (EXITS) genes, we examined somatic alterations from >4,100 cancers across 21 tumor types for sex bias. Six of 783 non-pseudoautosomal region (PAR) X-chromosome genes (ATRX, CNKSR2, DDX3X, KDM5C, KDM6A, and MAGEC3) harbored loss-of-function mutations more frequently in males (based on a false discovery rate < 0.1), in comparison to zero of 18,055 autosomal and PAR genes (Fisher's exact P < 0.0001). Male-biased mutations in genes that escape X-inactivation were observed in combined analysis across many cancers and in several individual tumor types, suggesting a generalized phenomenon. We conclude that biallelic expression of EXITS genes in females explains a portion of the reduced cancer incidence in females as compared to males across a variety of tumor types.

Mutations in the voltage-gated potassium channel gene KCNH1 cause Temple-Baraitser syndrome and epilepsy.

Temple-Baraitser syndrome (TBS) is a multisystem developmental disorder characterized by intellectual disability, epilepsy, and hypoplasia or aplasia of the nails of the thumb and great toe. Here we report damaging de novo mutations in KCNH1 (encoding a protein called ether à go-go, EAG1 or KV10.1), a voltage-gated potassium channel that is predominantly expressed in the central nervous system (CNS), in six individuals with TBS. Characterization of the mutant channels in both Xenopus laevis oocytes and human HEK293T cells showed a decreased threshold of activation and delayed deactivation, demonstrating that TBS-associated KCNH1 mutations lead to deleterious gain of function. Consistent with this result, we find that two mothers of children with TBS, who have epilepsy but are otherwise healthy, are low-level (10% and 27%) mosaic carriers of pathogenic KCNH1 mutations. Consistent with recent reports, this finding demonstrates that the etiology of many unresolved CNS disorders, including epilepsies, might be explained by pathogenic mosaic mutations.

Functional analysis of microRNAs in human hepatocellular cancer stem cells.

MicroRNAs are endogenous small non-coding RNAs that regulate gene expression and cancer development. A rare population of hepatocellular cancer stem cells (HSCs) holds the extensive proliferative and self-renewal potential necessary to form a liver tumour. We postulated that specific transcriptional factors might regulate the expression of microRNAs and subsequently modulate the expression of gene products involved in phenotypic characteristics of HSCs. We evaluated the expression of microRNA in human HSCs by microarray profiling, and defined the target genes and functional effects of two groups of microRNA regulated by IL-6 and transcriptional factor Twist. A subset of highly chemoresistant and invasive HSCs was screened with aberrant expressions of cytokine IL-6 and Twist. We demonstrated that conserved let-7 and miR-181 family members were up-regulated in HSCs by global microarray-based microRNA profiling followed by validation with real-time polymerase chain reaction. Importantly, inhibition of let-7 increases the chemosensitivity of HSCs to sorafenib and doxorubicin whereas silencing of miR-181 led to a reduction in HSCs motility and invasion. Knocking down IL-6 and Twist in HSCs significantly reduced let-7 and miR-181 expression and subsequently inhibited chemoresistance and cell invasion. We showed that let-7 directly targets SOCS-1 and caspase-3, whereas miR-181 directly targets RASSF1A, TIMP3 as well as nemo-like kinase (NLK). In conclusion, alterations of IL-6- and Twist-regulated microRNA expression in HSCs play a part in tumour spreading and responsiveness to chemotherapy. Our results define a novel regulatory mechanism of let-7/miR-181s suggesting that let-7 and miR-181 may be molecular targets for eradication of hepatocellular malignancies.

Optimized filtering reduces the error rate in detecting genomic variants by short-read sequencing.

Distinguishing single-nucleotide variants (SNVs) from errors in whole-genome sequences remains challenging. Here we describe a set of filters, together with a freely accessible software tool, that selectively reduce error rates and thereby facilitate variant detection in data from two short-read sequencing technologies, Complete Genomics and Illumina. By sequencing the nearly identical genomes from monozygotic twins and considering shared SNVs as 'true variants' and discordant SNVs as 'errors', we optimized thresholds for 12 individual filters and assessed which of the 1,048 filter combinations were effective in terms of sensitivity and specificity. Cumulative application of all effective filters reduced the error rate by 290-fold, facilitating the identification of genetic differences between monozygotic twins. We also applied an adapted, less stringent set of filters to reliably identify somatic mutations in a highly rearranged tumor and to identify variants in the NA19240 HapMap genome relative to a reference set of SNVs.

T. asahii pulmonary infection as a complication of TNF-inhibitor and steroids: posaconazole pharmacotherapy and risk analysis.

We report the first documented Trichosporon asahii infection in a patient with connective tissue disease treated with a Tumor Necrosis Factor (TNF) inhibitor and describe an institutional root cause analysis for TNF inhibitor-associated infections. Fourteen patients with incident fungal infections during TNF inhibitor treatment were identified. They were matched with uncomplicated patients receiving TNF inhibitors or with rheumatoid arthritis (RA) patients managed without TNF inhibitors. We found that patients acquiring fungal infections were more likely to have graft versus host disease (GVHD) (p<0.05). Furthermore, infected patients were more likely (OR=24.4) to have multiple immunosuppressive therapies over the controls as well as several risk factors identified by the Infectious Disease Society ofAmerica (IDSA). The 3 patient deaths in our study were associated with GVHD and infliximab. Trichosporon was isolated in 1 patient receiving adalimumab. Our results suggest that these high risk patients be monitored closely for fungal infection.

Mesentric oxygen saturations in premature twins with and without necrotizing enterocolitis.

OBJECTIVE: To report the use of near-infrared spectroscopic monitoring to recognize mesenteric oxygen desaturations in a preterm neonate with necrotizing enterocolitis as well as the demonstration of reassuring mesenteric tissue perfusion in a twin sibling with an uncomplicated course. DESIGN: Case report. SETTING: Neonatal intensive care unit in a tertiary care children's hospital. PATIENTS: A 12-day-old growth-restricted preterm female twin with necrotizing enterocolitis and her twin who did not develop disease. INTERVENTIONS: In the twin with symptoms of necrotizing enterocolitis, reduction in the mesenteric saturations was recorded in the injured bowel tissue as later confirmed during surgery. After resection of the ischemic bowel, mesenteric saturations returned to values comparable to those measured in the healthy twin. Reduced saturations were not observed in the asymptomatic twin. CONCLUSIONS: The use of optical oximetry to monitor mesenteric tissue saturation may provide a measure of bowel perfusion that could enhance clinical management in at-risk preterm neonates.

Clinical roundtable monograph: safety and efficacy of lipid-based amphotericin B.

Invasive fungal infections are an important cause of morbidity and mortality in critically ill and immunocompromised patients. Amphotericin B has remained the gold standard treatment for systemic fungal infections. The primary obstacle to amphotericin B therapy is its poor solubility and dose-related toxicities, especially renal impairment. As a result, newer lipid-based formulations of amphotericin B have been developed. This monograph compares available phamacokinetic, efficacy, and safety data for the 3 lipid-based formulations of amphotericin B, with reference to various patient populations. Potential causes of infusion-related reactions are described, and a premedication algorithm for the prevention of infusion-related reactions is developed.

CTCF cis-regulates trinucleotide repeat instability in an epigenetic manner: a novel basis for mutational hot spot determination.

At least 25 inherited disorders in humans result from microsatellite repeat expansion. Dramatic variation in repeat instability occurs at different disease loci and between different tissues; however, cis-elements and trans-factors regulating the instability process remain undefined. Genomic fragments from the human spinocerebellar ataxia type 7 (SCA7) locus, containing a highly unstable CAG tract, were previously introduced into mice to localize cis-acting "instability elements," and revealed that genomic context is required for repeat instability. The critical instability-inducing region contained binding sites for CTCF -- a regulatory factor implicated in genomic imprinting, chromatin remodeling, and DNA conformation change. To evaluate the role of CTCF in repeat instability, we derived transgenic mice carrying SCA7 genomic fragments with CTCF binding-site mutations. We found that CTCF binding-site mutation promotes triplet repeat instability both in the germ line and in somatic tissues, and that CpG methylation of CTCF binding sites can further destabilize triplet repeat expansions. As CTCF binding sites are associated with a number of highly unstable repeat loci, our findings suggest a novel basis for demarcation and regulation of mutational hot spots and implicate CTCF in the modulation of genetic repeat instability.

Effects of amphotericin B and caspofungin on histamine expression.

STUDY OBJECTIVE: To determine the effects of amphotericin B deoxycholate and caspofungin on the release of histamine from human peripheral blood cells, mononuclear cells, and mast cells. DESIGN: In vitro cell culture experiments. SETTING: University medical center. MATERIAL: Cultured human mononuclear (THP-1) and mast (HMC-1) cells from five healthy volunteers. MEASUREMENTS AND MAIN RESULTS: The cultured cells were incubated with increasing concentrations of amphotericin B deoxycholate, diphenhydramine, amphotericin B deoxycholate plus diphenhydramine, caspofungin, caspofungin plus diphenhydramine, and the calcium ionophore A23187 for up to 24 hours. Histamine concentrations and histamine N-methyltransferase activity were determined at various time points throughout exposure. Cell viability was assessed by exclusion of erythrocin B. The A23187 increased histamine concentrations from baseline in peripheral blood and HMC-1 cells. No change in histamine concentrations was observed in response to amphotericin B deoxycholate, whereas caspofungin induced a significant increase in histamine release in peripheral blood cells and HMC-1 cells. No change in histamine concentrations was observed in THP-1 cells in response to any pharmacologic agent tested. Similarly, histamine N-methyltransferase activity in peripheral blood was not affected by amphotericin B deoxycholate, but was significantly decreased by caspofungin. CONCLUSION: Amphotericin B deoxycholate does not affect histamine concentrations or histamine N-methyltransferase activity in whole blood or HMC-1 cells, suggesting that the amphotericin B-induced infusion-related reaction is not a histamine-mediated event. Conversely, caspofungin increased histamine concentrations in whole blood and HMC-1 cells and inhibited histamine N-methyltransferase activity. These data suggest that infusion-related reactions associated with caspofungin may be mediated by histamine release secondary to caspofungin.

Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cerevisiae.

Antifungal compounds exert their activity through a variety of mechanisms, some of which are poorly understood. Novel approaches to characterize the mechanism of action of antifungal agents will be of great use in the antifungal drug development process. The aim of the present study was to investigate the changes in the gene expression profile of Saccharomyces cerevisiae following exposure to representatives of the four currently available classes of antifungal agents used in the management of systemic fungal infections. Microarray analysis indicated differential expression of 0.8, 4.1, 3.0, and 2.6% of the genes represented on the Affymetrix S98 yeast gene array in response to ketoconazole, amphotericin B, caspofungin, and 5-fluorocytosine (5-FC), respectively. Quantitative real time reverse transcriptase-PCR was used to confirm the microarray analyses. Genes responsive to ketoconazole, caspofungin, and 5-FC were indicative of the drug-specific effects. Ketoconazole exposure primarily affected genes involved in ergosterol biosynthesis and sterol uptake; caspofungin exposure affected genes involved in cell wall integrity; and 5-FC affected genes involved in DNA and protein synthesis, DNA damage repair, and cell cycle control. In contrast, amphotericin B elicited changes in gene expression reflecting cell stress, membrane reconstruction, transport, phosphate uptake, and cell wall integrity. Genes with the greatest specificity for a particular drug were grouped together as drug-specific genes, whereas genes with a lack of drug specificity were also identified. Taken together, these data shed new light on the mechanisms of action of these classes of antifungal agents and demonstrate the potential utility of gene expression profiling in antifungal drug development.

Differential expression of genes encoding immunomodulatory proteins in response to amphotericin B in human mononuclear cells identified by cDNA microarray analysis.

Amphotericin B (AMB) is an antifungal agent that possesses immunomodulatory properties that may contribute to its infusion-related toxicity and activity. It has previously been shown to induce the expression of genes encoding the cytokines interleukin (IL)-1 beta and tumour necrosis factor (TNF)-alpha and the chemokines IL-8 and macrophage inflammatory protein (MIP)-1 beta in the human monocytic cell line THP-1. In an effort to identify additional AMB-responsive genes, the gene expression profiles of both THP-1 cells and human peripheral blood mononuclear cells (hPBMCs) on exposure to AMB were assessed using cDNA microarray analysis. In addition to genes known to be AMB responsive, we found the genes encoding IL-1 alpha and MIP-1 alpha to be AMB responsive in both THP-1 cells and hPBMCs. Increases in MIP-1 alpha and MIP-1 beta were also observed in the supernatants of hPBMCs exposed to AMB. The expression of several genes in response to AMB was unique to either cell type. Furthermore, variability in gene expression in hPBMCs was observed between donors. These genes and respective gene products may have significance in the infusion-related toxicity and activity of AMB.