Real-world effectiveness of antifungal prophylaxis with posaconazole as the primary agent in patients with haematological malignancies.

BACKGROUND AND OBJECTIVES: Patients undergoing induction/reinduction chemotherapy for haematologic malignancies (HM) are at risk for invasive fungal infections (IFIs). In 2015, Duke University Hospital (DUH) implemented a new standardised fungal prophylaxis protocol for adult patients undergoing induction chemotherapy for acute lymphocytic leukaemia, acute myelocytic leukaemia and myelodysplastic syndrome. This study assessed the impact of protocol implementation on (1) use of antifungal prophylaxis, throughout the at-risk period and (2) patient outcomes such as IFI and mortality. METHODS: Retrospective, observational study of adult HM patients admitted to DUH for induction/reinduction chemotherapy pre- (7/1/2013-12/31/2014) and post- (1/1/2015-10/31/2016) implementation of standardised antifungal prophylaxis protocol (which recommended posaconazole as the first-line agent). Patients were followed for up to 100 days after initiation of induction chemotherapy to evaluate use of antifungal prophylaxis and patient outcomes. RESULTS: 218 patients with haematologic malignancies were included (90 pre, 128 post). Use of antifungal prophylaxis increased from 81.1% (pre) to 97.7% (post) (p < .0001). Overall, 71% received posaconazole as initial antifungal prophylaxis (64.4% pre, 75.7% post). Approximately one-fourth of patients (25.6%, pre vs 26.6%, post) developed an IFI (proven/probable or possible using modified EORTC definitions) (p = .868); 100-day mortality remained stable (18.9% pre vs 18.8% post, respectively, p = .979). Lack of antifungal prophylaxis and older age (≥60 years) were associated with higher risk of IFI. CONCLUSION: Implementation of a standardised protocol with posaconazole as the primary agent was associated with increased use of antifungal prophylaxis among patients undergoing induction/reinduction chemotherapy for haematologic malignancies in our hospital. Lack of antifungal prophylaxis was an independent predictor of IFIs, underscoring the importance of prophylaxis in this at-risk population.

Trichodysplasia spinulosa: Case reports and review of literature.

Trichodysplasia spinulosa (TS) is a rare skin condition caused by trichodysplasia spinulosa-associated polyomavirus (TSPyV). It affects immunosuppressed patients, and <50 cases have been reported. The majority of these cases are seen in solid organ transplant recipients. TS often poses a diagnostic and therapeutic challenge because of its rarity and resemblance with other skin conditions. Several forms of treatment are usually tried prior to establishing a definitive diagnosis. Oral valganciclovir and topical cidofovir have been found to give the best results and hence are the most commonly used agents once the diagnosis is established. Here, we present two cases with a review of literature of TS in solid organ transplant recipients, focusing on time to develop the condition post-transplant, immunosuppression regimen used, and treatment initiated both before and after a definitive diagnosis.

A TfR-Binding Cystine-Dense Peptide Promotes Blood-Brain Barrier Penetration of Bioactive Molecules.

The impenetrability of the blood-brain barrier (BBB) to most conventional drugs impedes the treatment of central nervous system (CNS) disorders. Interventions for diseases like brain cancer, neurodegeneration, or age-associated inflammatory processes require varied approaches to CNS drug delivery. Cystine-dense peptides (CDPs) have drawn recent interest as drugs or drug-delivery vehicles. Found throughout the phylogenetic tree, often in drug-like roles, their size, stability, and protein interaction capabilities make CDPs an attractive mid-size biologic scaffold to complement conventional antibody-based drugs. Here, we describe the identification, maturation, characterization, and utilization of a CDP that binds to the transferrin receptor (TfR), a native receptor and BBB transporter for the iron chaperone transferrin. We developed variants with varying binding affinities (KD as low as 216 pM), co-crystallized it with the receptor, and confirmed murine cross-reactivity. It accumulates in the mouse CNS at ~25% of blood levels (CNS blood content is only ~1%-6%) and delivers neurotensin, an otherwise non-BBB-penetrant neuropeptide, at levels capable of modulating CREB signaling in the mouse brain. Our work highlights the utility of CDPs as a diverse, easy-to-screen scaffold family worthy of inclusion in modern drug discovery strategies, demonstrated by the discovery of a candidate CNS drug delivery vehicle ready for further optimization and preclinical development.

A potent peptide-steroid conjugate accumulates in cartilage and reverses arthritis without evidence of systemic corticosteroid exposure.

On-target, off-tissue toxicity limits the systemic use of drugs that would otherwise reduce symptoms or reverse the damage of arthritic diseases, leaving millions of patients in pain and with limited physical mobility. We identified cystine-dense peptides (CDPs) that rapidly accumulate in cartilage of the knees, ankles, hips, shoulders, and intervertebral discs after systemic administration. These CDPs could be used to concentrate arthritis drugs in joints. A cartilage-accumulating peptide, CDP-11R, reached peak concentration in cartilage within 30 min after administration and remained detectable for more than 4 days. Structural analysis of the peptides by crystallography revealed that the distribution of positive charge may be a distinguishing feature of joint-accumulating CDPs. In addition, quantitative whole-body autoradiography showed that the disulfide-bonded tertiary structure is critical for cartilage accumulation and retention. CDP-11R distributed to joints while carrying a fluorophore imaging agent or one of two different steroid payloads, dexamethasone (dex) and triamcinolone acetonide (TAA). Of the two payloads, the dex conjugate did not advance because the free drug released into circulation was sufficient to cause on-target toxicity. In contrast, the CDP-11R-TAA conjugate alleviated joint inflammation in the rat collagen-induced model of rheumatoid arthritis while avoiding toxicities that occurred with nontargeted steroid treatment at the same molar dose. This conjugate shows promise for clinical development and establishes proof of concept for multijoint targeting of disease-modifying therapeutic payloads.

Use of Letermovir as Salvage Therapy for Drug-Resistant Cytomegalovirus Retinitis.

Treatment options for drug-resistant cytomegalovirus (CMV) are limited. Letermovir is a novel antiviral recently approved for CMV prophylaxis following hematopoietic cell transplantation, but its efficacy in other settings is unknown. We recently used letermovir for salvage treatment in four solid organ transplant recipients with ganciclovir-resistant CMV retinitis. All patients improved clinically without known adverse drug events. However, three patients failed to maintain virologic suppression, including two patients who developed genotypically confirmed resistance to letermovir while on therapy.

A biobank of patient-derived pediatric brain tumor models.

Brain tumors are the leading cause of cancer-related death in children. Genomic studies have provided insights into molecular subgroups and oncogenic drivers of pediatric brain tumors that may lead to novel therapeutic strategies. To evaluate new treatments, better preclinical models adequately reflecting the biological heterogeneity are needed. Through the Children's Oncology Group ACNS02B3 study, we have generated and comprehensively characterized 30 patient-derived orthotopic xenograft models and seven cell lines representing 14 molecular subgroups of pediatric brain tumors. Patient-derived orthotopic xenograft models were found to be representative of the human tumors they were derived from in terms of histology, immunohistochemistry, gene expression, DNA methylation, copy number, and mutational profiles. In vivo drug sensitivity of targeted therapeutics was associated with distinct molecular tumor subgroups and specific genetic alterations. These models and their molecular characterization provide an unprecedented resource for the cancer community to study key oncogenic drivers and to evaluate novel treatment strategies.

A technology platform to assess multiple cancer agents simultaneously within a patient's tumor.

A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

Distinct neuroinflammatory profile in post-mortem human Huntington's disease.

Neuroinflammation is a prominent feature of many neurodegenerative diseases, however, little is known about neuroinflammation in Huntington's disease. We used quantitative real time-PCR to compare the expression level of neuroinflammation-associated mediators in the striatum, cortex, and cerebellum from post-mortem Huntington's disease patient samples with controls. We found increased expression of several key inflammatory mediators, including CCL2 and IL-10, specifically in the striatum of Huntington's disease patients, the main area affected by this pathology. Remarkably, we also found upregulation of IL-6, IL-8, and MMP9, in the cortex and notably the cerebellum, a brain area commonly thought to be spared by Huntington's disease. Our data suggest that neuroinflammation is a prominent feature associated with Huntington's disease and may constitute a novel target for therapeutic intervention.

Huntingtin interacting proteins are genetic modifiers of neurodegeneration.

Huntington's disease (HD) is a fatal neurodegenerative condition caused by expansion of the polyglutamine tract in the huntingtin (Htt) protein. Neuronal toxicity in HD is thought to be, at least in part, a consequence of protein interactions involving mutant Htt. We therefore hypothesized that genetic modifiers of HD neurodegeneration should be enriched among Htt protein interactors. To test this idea, we identified a comprehensive set of Htt interactors using two complementary approaches: high-throughput yeast two-hybrid screening and affinity pull down followed by mass spectrometry. This effort led to the identification of 234 high-confidence Htt-associated proteins, 104 of which were found with the yeast method and 130 with the pull downs. We then tested an arbitrary set of 60 genes encoding interacting proteins for their ability to behave as genetic modifiers of neurodegeneration in a Drosophila model of HD. This high-content validation assay showed that 27 of 60 orthologs tested were high-confidence genetic modifiers, as modification was observed with more than one allele. The 45% hit rate for genetic modifiers seen among the interactors is an order of magnitude higher than the 1%-4% typically observed in unbiased genetic screens. Genetic modifiers were similarly represented among proteins discovered using yeast two-hybrid and pull-down/mass spectrometry methods, supporting the notion that these complementary technologies are equally useful in identifying biologically relevant proteins. Interacting proteins confirmed as modifiers of the neurodegeneration phenotype represent a diverse array of biological functions, including synaptic transmission, cytoskeletal organization, signal transduction, and transcription. Among the modifiers were 17 loss-of-function suppressors of neurodegeneration, which can be considered potential targets for therapeutic intervention. Finally, we show that seven interacting proteins from among 11 tested were able to co-immunoprecipitate with full-length Htt from mouse brain. These studies demonstrate that high-throughput screening for protein interactions combined with genetic validation in a model organism is a powerful approach for identifying novel candidate modifiers of polyglutamine toxicity.

Contribution of nuclear and extranuclear polyQ to neurological phenotypes in mouse models of Huntington's disease.

In postmortem Huntington's disease brains, mutant htt is present in both nuclear and cytoplasmic compartments. To dissect the impact of nuclear and extranuclear mutant htt on the initiation and progression of disease, we generated a series of transgenic mouse lines in which nuclear localization or nuclear export signal sequences have been placed N-terminal to the htt exon 1 protein carrying 144 glutamines. Our data indicate that the exon 1 mutant protein is present in the nucleus as part of an oligomeric or aggregation complex. Increasing the concentration of the mutant transprotein in the nucleus is sufficient for and dramatically accelerates the onset and progression of behavioral phenotypes. Furthermore, nuclear exon 1 mutant protein is sufficient to induce cytoplasmic neurodegeneration and transcriptional dysregulation. However, our data suggest that cytoplasmic mutant exon 1 htt, if present, contributes to disease progression.

Genetic heterogeneity of stably transfected cell lines revealed by expression profiling with oligonucleotide microarrays.

Large-scale gene expression measurements with oligonucleotide microarrays have contributed tremendously to biological research. However, to distinguish between relevant expression changes and falsely identified positives, the source and magnitude of errors must be understood. Here, we report a source of biological variability in microarray experiments with stably transfected cell lines. Mouse embryonic fibroblast (MEF/3T3) and rat schwannoma (RT4) cell lines were generated to provide regulatable schwannomin expression. The expression levels of 29 samples from five different mouse embryonic fibroblast clonal cell lines and 18 samples from 3 RT4 cell lines were monitored with oligonucleotide microarrays. Using hierarchical clustering, we determined that the changes in gene expression induced by schwannomin overexpression were subtle when compared with those detected as a consequence of clonal selection during generation of the cell lines. The hierarchical clustering implies that significant alterations of gene expression were introduced during the transfection and selection processes. A total of 28 genes were identified by Kruskal-Wallis rank test that showed significant variation between clonal lines. Most of them were related to cytoskeletal function and signaling pathways. Based on these analyses, we recommend that replications of experiments with several selected cell lines are necessary to assess biological effects of induced gene expression.

BMP-2 mediates retinoid-induced apoptosis in medulloblastoma cells through a paracrine effect.

The mechanisms of retinoid activity in tumors remain largely unknown. Here we establish that retinoids cause extensive apoptosis of medulloblastoma cells. In a xenograft model, retinoids largely abrogated tumor growth. Using receptor-specific retinoid agonists, we defined a subset of mRNAs that were induced by all active retinoids in retinoid-sensitive cell lines. We also identified bone morphogenetic protein-2 (BMP-2) as a candidate mediator of retinoid activity. BMP-2 protein induced medulloblastoma cell apoptosis, whereas the BMP-2 antagonist noggin blocked both retinoid and BMP-2-induced apoptosis. BMP-2 also induced p38 mitogen-activated protein kinase (MAPK), which is necessary for BMP-2- and retinoid-induced apoptosis. Retinoid-resistant medulloblastoma cells underwent apoptosis when treated with BMP-2 or when cultured with retinoid-sensitive medulloblastoma cells. Retinoid-induced expression of BMP-2 is thus necessary and sufficient for apoptosis of retinoid-responsive cells, and expression of BMP-2 by retinoid-sensitive cells is sufficient to induce apoptosis in surrounding retinoid-resistant cells.

Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain.

Previous analyses of gene expression in a mouse model of Huntington's disease (R6/2) indicated that an N-terminal fragment of mutant huntingtin causes downregulation of striatal signaling genes and particularly those normally induced by cAMP and retinoic acid. The present study expands the regional and temporal scope of this previous work by assessing whether similar changes occur in other brain regions affected in Huntington's disease and other polyglutamine diseases and by discerning whether gene expression changes precede the appearance of disease signs. Oligonucleotide microarrays were employed to survey the expression of approximately 11,000 mRNAs in the cerebral cortex, cerebellum and striatum of symptomatic R6/2 mice. The number and nature of gene expression changes were similar among these three regions, influenced as expected by regional differences in baseline gene expression. Time-course studies revealed that mRNA changes could only reliably be detected after 4 weeks of age, coincident with development of early pathologic and behavioral changes in these animals. In addition, we discovered that skeletal muscle is also a target of polyglutamine-related perturbations in gene expression, showing changes in mRNAs that are dysregulated in brain and also muscle-specific mRNAs. The complete dataset is available at www.neumetrix.info.

Polyglutamine and transcription: gene expression changes shared by DRPLA and Huntington's disease mouse models reveal context-independent effects.

Recent evidence indicates that transcriptional abnormalities may play an important role in the pathophysiology of polyglutamine diseases. In the present study, we have explored the extent to which polyglutamine-related changes in gene expression may be independent of protein context by comparing mouse models of dentatorubral-pallidoluysian atrophy (DRPLA) and Huntington's disease (HD). Microarray gene expression profiling was conducted in mice of the same background strain in which the same promoter was employed to direct the expression of full-length atrophin-1 or partial huntingtin transproteins (At-65Q or N171-82Q mice). A large number of overlapping gene expression changes were observed in the cerebella of At-65Q and N171-82Q mice. Six of the gene expression changes common to both huntingtin and atrophin-1 transgenic mice were also observed in the cerebella of mouse models expressing full-length mutant ataxin-7 or the androgen receptor. These results demonstrate that some of the gene expression effects of expanded polyglutamine proteins occur independently of protein context.

Increased huntingtin protein length reduces the number of polyglutamine-induced gene expression changes in mouse models of Huntington's disease.

Both transcriptional dysregulation and proteolysis of mutant huntingtin (htt) are postulated to be important components of Huntington's disease (HD) pathogenesis. In previous studies, we demonstrated that transgenic mice that express short mutant htt fragments containing 171 or fewer N-terminal residues (R6/2 and N171-82Q mice) recapitulate many of the mRNA changes observed in human HD brain. To examine whether htt protein length influences the ability of its expanded polyglutamine domain to alter gene expression, we conducted mRNA profiling analyses of mice that express an extended N-terminal fragment (HD46, HD100; 964 amino acids) or full-length (YAC72; 3144 amino acids) mutant htt transprotein. Oligonucleotide microarray analyses of HD46 and YAC72 mice identified fewer differentially expressed mRNAs than were seen in transgenic mice expressing short N-terminal mutant htt fragments. Histologic analyses also detected limited changes in these mice (small decreases in adenosine A2a receptor mRNA and dopamine D2 receptor binding in HD100 animals; small increases in dopamine D1 receptor binding in HD46 and HD100 mice). Neither HD46 nor YAC72 mice exhibited altered mRNA levels similar to those observed previously in R6/2 mice, N171-82Q mice or human HD patients. These findings suggest that htt protein length influences the ability of an expanded polyglutamine domain to alter gene expression. Furthermore, our findings suggest that short N-terminal fragments of mutant htt might be responsible for the gene expression alterations observed in human HD brain.

Altered transcriptional regulation in cells expressing the expanded polyglutamine androgen receptor.

Kennedy's disease is a degenerative disease of motor neurons in which the causative mutation is expansion of a CAG/polyglutamine tract near the 5' end of the androgen receptor gene. The mutant protein misfolds, aggregates, and interacts abnormally with other proteins, leading to a novel, toxic gain of function and an alteration of normal function. We used a cell culture model to explore the mechanisms underlying the alterations in androgen receptor function conferred by the mutation. Here we show that cells expressing the wild-type androgen receptor with 24 CAG repeats respond to ligand by showing trophic effects including prolonged survival in low serum, whereas cells expressing the mutant receptor with 65 CAG repeats do not show a robust trophic response. This partial loss of function correlates with decreased levels of the mutant protein due to its preferential degradation by the ubiquitin-proteasome pathway. Expression analysis using oligonucleotide arrays confirms that the mutant receptor has undergone a partial loss of function, and fails to regulate a subset of genes whose expression is normally affected by ligand activation of the wild-type receptor. The mutant receptor has also undergone several functionally important post-translational modifications in the absence of ligand that the wild-type receptor undergoes in the presence of ligand, including acetylation and phosphorylation. These modifications correlate with a ligand-independent gain of function exhibited by the mutant receptor in expression analysis. Our findings suggest that polyglutamine expansion alters androgen receptor function by promoting its degradation and by modifying its activity as a transcription factor.