Pharmacokinetic analysis driven letermovir dosing in pediatric hematopoietic cell transplantation patients with resistant CMV disease.

BACKGROUND: Reactivation of cytomegalovirus (CMV) in CMV-seropositive patients after haploidentical T-cell receptor αß+ /CD19+ depleted hematopoietic cell transplant (HCT) is common. Due to delayed CMV-specific immune reconstitution, patients may require prolonged antiviral therapy, including secondary prophylaxis (SP). We present our clinical experience with the off-label use of letermovir for SP in a severely immunocompromised 2-year-old toddler with refractory pre-B-cell ALL and bilateral retinitis caused by resistant CMV (A594V UL97 mutation) following a haploidentical TCRαß+ /CD19+ depleted HCT. METHODS: The patient underwent measurement of two separate sets of letermovir serum concentrations, drawn at pre-dose, 1 and 4 h (and 8 h during the second therapeutic drug monitoring) post-dose. Pharmacokinetic parameters, including AUC0-24 were calculated, and dose adjustment was performed based on the drug level. RESULTS: While receiving oral letermovir 240 mg once daily without cyclosporine, the observed AUC0-24 was high (75 815 ng h/mL) with a Cmin of 209 ng/mL. The dose was reduced by 25% to 180 mg once daily. Despite the dose reduction, both AUC0-24 and Cmin values further increased to 119 095 ng h/L and 959 ng/mL, respectively. The patient continued oral letermovir 180 mg once daily for about 3 months, with adequate viral suppression (CMV viral load in plasma <150 IU/mL) and no recurrent CMV end-organ disease or adverse events. CONCLUSIONS: Given limited options for anti-CMV therapy in young children with resistant CMV, letermovir could be considered as an alternative antiviral for SP. Further studies are warranted to evaluate the pharmacokinetics of letermovir in pediatric allogeneic HCT recipients.

Additive effect of walnut and chokeberry on regulation of antioxidant enzyme gene expression and attenuation of lipid peroxidation in d-galactose-induced aging-mouse model.

Studies have highlighted the association between the cellular damage caused by reactive oxygen species and aging. The reducing sugar d-galactose causes aging-related changes and oxidative stress. Lipids are the first target of free radicals, and lipid peroxidation is related to aging. Walnut (Juglans regia Chandler) kernel contains antioxidant phenolic compounds, and chokeberry (Aronia melanocarpa) is one of the richest sources of polyphenols, including anthocyanins, among other fruits. Polyphenols from chokeberry exhibit antioxidant and anti-inflammatory activities. In this study, the additive antioxidative effect of walnut and chokeberry mixture was evaluated by oxidative stress index in d-galactose-induced aging model. Thirty-five Balb/c mice (8 weeks old) were divided into following five groups (n = 7 in each group): normal control (C), d-galactose control (D), d-galactose with chokeberry diet (CH), d-galactose with walnut diet (W), and d-galactose with walnut and chokeberry mixture diet (WCH). In all treatment diets groups, the levels of serum, hepatic, and kidney malonaldehyde were significantly lower than D group and the levels were approaching to control level. Moreover, the kidney malondialdehyde levels were significantly lower in WCH group compared with the control group. This study also confirmed the activities of antioxidant enzymes in liver, as the levels of superoxide dismutase, and glutathione peroxidase were significantly increased in CH group compared to in W or CH groups. The results of this study supported the additive effect of walnut and chokeberry on increment of antioxidant enzyme gene expression in liver and consequently the attenuation of lipid peroxidation in serum, liver, and kidney in d-galactose-induced aging-mouse model. Further studies are needed to investigate the detailed mechanism underlying the additive antioxidative effects in various tissues.

Acanthamoeba granulomatous amoebic encephalitis after pediatric hematopoietic stem cell transplant.

Acanthamoeba encephalitis is a rare, often fatal condition, particularly after HSCT, with 9 reported cases to date in the world literature. Our case was originally diagnosed with ALL at age 3 years, and after several relapses underwent HSCT at age 9 years. At 17 years of age, he was diagnosed with secondary AML for which he underwent a second allogeneic HSCT. He presented with acute-onset worsening neurological deficits on day +226 after the second transplant and a post-mortem diagnosis of Acanthamoeba encephalitis was established, with the aid of the CDC.

Adenovirus and "Culture-Negative Sepsis" in a Preterm Neonate.

Background Respiratory viral infections remain an underrecognized cause of morbidity and mortality among preterm infants in the neonatal intensive care unit (NICU). Case Report An eight day old, 650 gram birth weight, 23 weeks' gestational age female developed "culture-negative" sepsis manifested by respiratory deterioration, hypoxia, leukocytosis, and thrombocytopenia. She was diagnosed with pneumonia and hepatitis due to adenovirus HAdV-D (H29F9) by polymerase chain reaction (PCR) testing, but died at the age of 18 days despite treatment with cidofovir and immune globulin intravenous. Conclusion As the ability to diagnosis respiratory viral infections in the NICU has improved greatly with the use of PCR testing, the impact and contribution of these viruses to neonatal disease is now being recognized and the notion of "culture-negative" sepsis needs reassessment. The diagnosis of these infections in high risk infants is important not only for etiologic and epidemiologic reasons but ultimately for informing antimicrobial stewardship efforts.

A protracted course of Pneumocystis pneumonia in the setting of an immunosuppressed child with GMS-negative bronchoalveolar lavage.

We report a case of Pneumocystis pneumonia in a 5-year-old male with Trisomy 21 and acute lymphoblastic leukemia. The lack of response to trimethoprim-sulfamethoxazole raised concerns for antimicrobial resistance. Further, diagnosis of Pneumocystis in this patient was complicated by a GMS-negative bronchoalveolar lavage despite molecular evidence of Pneumocystis infection.

Performance Characteristics of FilmArray Respiratory Panel v1.7 for Detection of Adenovirus in a Large Cohort of Pediatric Nasopharyngeal Samples: One Test May Not Fit All.

The FilmArray Respiratory Panel (RP) v1.7 assay has improved sensitivity for detection of human adenovirus (HAdV), compared to an earlier version (RP v1.6). RP v1.7 was designed for detection of species B, C, and E but may show variable detection of species A, D, and F. We sought to evaluate the clinical and analytical performance of RP v1.7 for detection of HAdV in a large pediatric cohort. Respiratory specimens obtained from a tertiary care children's hospital between February 2014 and February 2015 were tested for HAdV by RP v1.7. If the RP v1.7 results were negative for HAdV, then the specimens were reflexed to a HAdV-specific laboratory-developed PCR (LD-PCR) assay for confirmation. A subset of specimens underwent secondary confirmatory testing using another commercially available HAdV PCR assay and a molecular typing assay for species identification. Among 4,750 specimens, a total of 146 specimens (3.1%) were HAdV positive by RP v1.7. HAdV was detected by LD-PCR in an additional 220 specimens that were negative by RP v1.7. Overall, a nearly 5% increase in HAdV detection was observed when RP v1.7-negative specimens were reflexed to LD-PCR testing. RP v1.7 did not detect HAdV with either low viral burden (threshold cycle values of >30) or nonrespiratory species (species A, D, and F), as shown in both clinical and analytic data. While the level of sensitivity of RP v1.7 may be adequate for testing among otherwise healthy children, the decreased sensitivity may be problematic for immunocompromised patients, in whom low levels of HAdV in the respiratory tract may precede systemic infection and require early intervention.

Diagnosis of Pediatric Acute Adenovirus Infections: Is a Positive PCR Sufficient?

BACKGROUND: Human adenovirus (HAdV), especially species C (HAdV-C), can be detected incidentally by polymerase chain reaction in nasopharyngeal (NP) samples, making it difficult to interpret clinical significance of a positive result. We classified patients into groups based on HAdV culture positivity from respiratory specimens and the presence of an identified co-pathogen. We hypothesized that HAdV-C would be over-represented and viral burden would be lower in patients most likely to have incidental detection (ie, with a negative viral culture and documented co-pathogen). METHODS: Immunocompetent children with HAdV + nasopharyngeal specimens were classified into 4 groups: group I (HAdV culture (+) and no co-infection), group II (culture (+) and co-infection), group III (culture (-) and no co-infection) and group IV (culture (-) and co-infection). Viral burden (cycle threshold) and species were compared among groups. RESULTS: Of 483 nasopharyngeal specimens, HAdV was isolated in culture in 252 (52%); co-infection was found in 265 (55%) patients. Group I (most consistent with acute disease) had significantly lower cycle thresholds (median 23.9; interquarile range 22.2-28.1) compared with group IV (most consistent with incidental detection; median 37.3; interquarile range 35.3-38.9; P < 0.0001). HAdV-C accounted for 41% samples of group I and 83% of group IV (P < 0.0001). We identified a subset of 22 patients with bacterial or fungal co-pathogens, 18 of whom had no growth on viral culture (group IV) with a median cycle threshold of 37.4 (interquarile range 33.9-39.2). CONCLUSIONS: Species identification and viral burden may assist in interpretation of a positive HAdV result. Low viral burden with HAdV-C may be consistent with incidental detection.

Clinical and Virologic Characteristics May Aid Distinction of Acute Adenovirus Disease from Kawasaki Disease with Incidental Adenovirus Detection.

Incidental adenovirus detection in Kawasaki disease (KD) is important to differentiate from acute adenovirus disease. Twenty-four of 25 children with adenovirus disease and mimicking features of KD had <4 KD-like features, predominance of species B or E, and higher viral burden compared with those with KD and incidental adenovirus detection.

Interferon ß protects against lethal endotoxic and septic shock through SIRT1 upregulation.

Lipopolysaccharide (LPS), an endotoxin derived from gram-negative bacteria, promotes the secretion of proinflammatory cytokines and mediates endotoxemia through activation of mitogen activated protein kinases, NF-κB, and interferon regulatory factor-3. Silent information regulator transcript-1 (SIRT1), an NAD-dependent deacetylase, mediates NF-κB deacetylation, and inhibits its function. SIRT1 may affect LPS-mediated signaling pathways and endotoxemia. Here we demonstrate that SIRT1 blocks LPS-induced secretion of interleukin 6 and tumor necrosis factor α in murine macrophages, and protects against lethal endotoxic and septic shock in mice. We also demonstrate that interferon ß increases SIRT1 expression by activating the Janus kinase--signal transducer and activator of transcription (JAK-STAT) pathway in mouse bone marrow derived macrophages. In vivo treatment of interferon ß protects against lethal endotoxic and septic shock, which is abrogated by infection with dominant negative SIRT1-expressing adenovirus. Our work suggests that both SIRT1 and SIRT1-inducing cytokines are useful targets for treating patients with sepsis.

Interplay between estrogen receptor and AKT in estradiol-induced alternative splicing.

BACKGROUND: Alternative splicing is critical for generating complex proteomes in response to extracellular signals. Nuclear receptors including estrogen receptor alpha (ERα) and their ligands promote alternative splicing. The endogenous targets of ERα:estradiol (E2)-mediated alternative splicing and the influence of extracellular kinases that phosphorylate ERα on E2-induced splicing are unknown. METHODS: MCF-7 and its anti-estrogen derivatives were used for the majority of the assays. CD44 mini gene was used to measure the effect of E2 and AKT on alternative splicing. ExonHit array analysis was performed to identify E2 and AKT-regulated endogenous alternatively spliced apoptosis-related genes. Quantitative reverse transcription polymerase chain reaction was performed to verify alternative splicing. ERα binding to alternatively spliced genes was verified by chromatin immunoprecipitation assay. Bromodeoxyuridine incorporation-ELISA and Annexin V labeling assays were done to measure cell proliferation and apoptosis, respectively. RESULTS: We identified the targets of E2-induced alternative splicing and deconstructed some of the mechanisms surrounding E2-induced splicing by combining splice array with ERα cistrome and gene expression array. E2-induced alternatively spliced genes fall into at least two subgroups: coupled to E2-regulated transcription and ERα binding to the gene without an effect on rate of transcription. Further, AKT, which phosphorylates both ERα and splicing factors, influenced ERα:E2 dependent splicing in a gene-specific manner. Genes that are alternatively spliced include FAS/CD95, FGFR2, and AXIN-1. E2 increased the expression of FGFR2 C1 isoform but reduced C3 isoform at mRNA level. E2-induced alternative splicing of FAS and FGFR2 in MCF-7 cells correlated with resistance to FAS activation-induced apoptosis and response to keratinocyte growth factor (KGF), respectively. Resistance of MCF-7 breast cancer cells to the anti-estrogen tamoxifen was associated with ERα-dependent overexpression of FGFR2, whereas resistance to fulvestrant was associated with ERα-dependent isoform switching, which correlated with altered response to KGF. CONCLUSION: E2 may partly alter cellular proteome through alternative splicing uncoupled to its effects on transcription initiation and aberration in E2-induced alternative splicing events may influence response to anti-estrogens.

Lipopolysaccharide induces CD38 expression and solubilization in J774 macrophage cells.

CD38, an ADP ribosyl cyclase, is a 45 kDa type II transmembrane protein having a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain, expressed on the surface of various cells including macrophages, lymphocytes, and pancreatic ß cells. It is known to be involved in cell adhesion, signal transduction and calcium signaling. In addition to its transmembrane form, CD38 is detectable in biological fluids in soluble forms. The mechanism by which CD38 is solubilized from the plasma membrane is not yet clarified. In this study, we found that lipopolysaccharide (LPS) induced CD38 upregulation and its extracellular release in J774 macrophage cells. Furthermore, it also increased CD38 expression at the mRNA level by activating the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. However, LPS decreased the levels of CD38 in the plasma membrane by releasing CD38 into the culture supernatant. LPS-induced CD38 release was blocked by the metalloproteinase-9 inhibitor indicating that MMP-9 solubilizes CD38. In conclusion, the present findings demonstrate a potential mechanism by which C38 is solubilized from the plasma membrane.

NAADP mediates insulin-stimulated glucose uptake and insulin sensitization by PPARγ in adipocytes.

Insulin stimulates glucose uptake through the membrane translocation of GLUT4 and GLUT1. Peroxisome proliferator-activated receptor γ (PPARγ) enhances insulin sensitivity. Here, we demonstrate that insulin stimulates GLUT4 and GLUT1 translocation, and glucose uptake, by activating the signaling pathway involving nicotinic acid adenine dinucleotide phosphate (NAADP), a calcium mobilizer, in adipocytes. We also demonstrate that PPARγ mediates insulin sensitization by enhancing NAADP production through upregulation of CD38, the only enzyme identified for NAADP synthesis. Insulin produced NAADP by both CD38-dependent and -independent pathways, whereas PPARγ produced NAADP by CD38-dependent pathway. Blocking the NAADP signaling pathway abrogated both insulin-stimulated and PPARγ-induced GLUT4 and GLUT1 translocation, thereby inhibiting glucose uptake. CD38 knockout partially inhibited insulin-stimulated glucose uptake. However, CD38 knockout completely blocked PPARγ-induced glucose uptake in adipocytes and PPARγ-mediated amelioration of glucose tolerance in diabetic mice. These results demonstrated that the NAADP signaling pathway is a critical molecular target for PPARγ-mediated insulin sensitization.

Concomitant gene mutations of MBL and CYBB in chronic granulomatous disease: implications for host defense.

Chronic granulomatous disease (CGD) is associated with defective function of the NADPH-oxidase system in conjunction with phagocytic defects which leads to granuloma formation and serious infectious complications. This is often associated with significant morbidity and mortality. The association of defective phagocyte function with other coincidental immune defects is unknown. Defects in innate pathways seen with CGD, including complement systems, and toll-like and dectin receptor pathways, have not been described before. We present the case of a 2-year old male patient hospitalized with recurrent pneumonia, a non-healing skin ulcer, necrotizing lung granulomas, and epididymo-orchitis. Defective neutrophil chemiluminescence was detected by dihydrorhodamine (DHR) testing. Further evaluation demonstrated characteristic molecular mutations of CYBB consistent with CGD. Immune evaluation demonstrated polyclonal hyperglobulinemia, but a greatly reduced mannose binding lectin (MBL) level. Six biallelic polymorphisms in MBL gene and its promoter were analyzed using Light CyclerTM Real-time PCR assay. The LXPA/LYPB haplotype of MBL was detected in our patient; the latter is the defective haplotype associated with low MBL levels. Due to the implications for innate immunity and the protection against bacterial, viral, and fungal infections provided by MBL, a deficiency of this protein may have disastrous consequences on the long term outcomes of CGD. MBL deficiency can also complicate other disorders affecting the immune system, significantly increasing the risk of infection in such patients. Further studies looking at the frequency and implications of MBL deficiency in CGD are needed.

SIRT1 Inhibits p53 but not NF-κB Transcriptional Activity during Differentiation of Mouse Embryonic Stem Cells into Embryoid Bodies.

BACKGROUND AND OBJECTIVES: SIRT1, a histone diacetylase, modify transactivation function of various transcription factor including p53 and NF-κB. p53 and NF-κB is involved in in vitro differentiation of mouse embryonic stem cells (mESC) into mouse embryoid body (mEB). These suggest that SIRT1 might affect in vitro differentiation of mESC into mEB by regulation of p53 and NF-κB. METHODS AND RESULTS: In this study we analyzed the effect of SIRT1 in in vitro differentiation of mESC into mEB using wild and SIRT1 knockout mESC. To examine SIRT1-specific gene in mESC, this study conducted microarray-based differential gene expression analysis between wild and SIRT1 knockout mESC. Comparing their gene expression patterns, this study determined a list of genes regulated by SIRT1. cDNA microarray data-set analysis revealed that genes associated with transcription and signal transduction are significantly modified in SIRT1 knockout mESC. cDNA microarray data-set analysis between mESC and EB in wild and SIRT1 showed that SIRT1 inhibits p53 signaling pathway but not affect NF-κB signaling pathway. CONCLUSIONS: This study suggests that SIRT1 modify mESC differentiation by regulation of p53 transcriptional activity.

Chronic granulomatous disease, the McLeod phenotype and the contiguous gene deletion syndrome-a review.

Chronic Granulomatous Disease (CGD), a disorder of the NADPH oxidase system, results in phagocyte functional defects and subsequent infections with bacterial and fungal pathogens (such as Aspergillus species and Candida albicans). Deletions and missense, frameshift, or nonsense mutations in the gp91phox gene (also termed CYBB), located in the Xp21.1 region of the X chromosome, are associated with the most common form of CGD. When larger X-chromosomal deletions occur, including the XK gene deletion, a so-called "Contiguous Gene Deletion Syndrome" may result. The contiguous gene deletion syndrome is known to associate the Kell phenotype/McLeod syndrome with diseases such as X-linked chronic granulomatous disease, Duchenne muscular dystrophy, and X-linked retinitis pigmentosa. These patients are often complicated and management requires special attention to the various facets of the syndrome.

Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport.

The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. However, it is unclear how cADPR extracellularly generated by ecto-CD38 approaches intracellular RyR although CD38 itself or nucleoside transporter has been proposed to import cADPR. Moreover, it has been unknown what physiological stimulation can trigger Cx43-mediated export of NAD(+). Here we demonstrate that Cx43 hemichannels, but not CD38, import cADPR to increase intracellular calcium through RyR. We also demonstrate that physiological stimulation such as Fcγ receptor (FcγR) ligation induces calcium mobilization through three sequential steps, Cx43-mediated NAD(+) export, CD38-mediated generation of cADPR and Cx43-mediated cADPR import in J774 cells. Protein kinase A (PKA) activation also induced calcium mobilization in the same way as FcγR stimulation. FcγR stimulation-induced calcium mobilization was blocked by PKA inhibition, indicating that PKA is a linker between FcγR stimulation and NAD(+)/cADPR transport. Cx43 knockdown blocked extracellular cADPR import and extracellular cADPR-induced calcium mobilization in J774 cells. Cx43 overexpression in Cx43-negative cells conferred extracellular cADPR-induced calcium mobilization by the mediation of cADPR import. Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.

Chronic granulomatous disease: a review of the infectious and inflammatory complications.

Chronic Granulomatous Disease is the most commonly encountered immunodeficiency involving the phagocyte, and is characterized by repeated infections with bacterial and fungal pathogens, as well as the formation of granulomas in tissue. The disease is the result of a disorder of the NADPH oxidase system, culminating in an inability of the phagocyte to generate superoxide, leading to the defective killing of pathogenic organisms. This can lead to infections with Staphylococcus aureus, Psedomonas species, Nocardia species, and fungi (such as Aspergillus species and Candida albicans). Involvement of vital or large organs can contribute to morbidity and/or mortality in the affected patients. Major advances have occurred in the diagnosis and treatment of this disease, with the potential for gene therapy or stem cell transplantation looming on the horizon.

Overexpression of SIRT1 protects pancreatic beta-cells against cytokine toxicity by suppressing the nuclear factor-kappaB signaling pathway.

OBJECTIVE: SIRT1, a class III histone/protein deacetylase, is known to interfere with the nuclear factor-kappaB (NF-kappaB) signaling pathway and thereby has an anti-inflammatory function. Because of the central role of NF-kappaB in cytokine-mediated pancreatic beta-cell damage, we postulated that SIRT1 might work in pancreatic beta-cell damage models. RESEARCH DESIGN AND METHODS: RINm5F (RIN) cells or isolated rat islets were treated with interleukin-1beta and interferon-gamma. SIRT1 was activated by resveratrol, a pharmacological activator, or ectopic overexpression. The underlying mechanisms of SIRT1 against cytokine toxicity were further explored. RESULTS: Treatment of RIN cells with cytokines induced cell damage, and this damage was well correlated with the expression of the inducible form of nitric oxide (NO) synthase (iNOS) and NO production. However, SIRT1 overexpression completely prevented cytokine-mediated cytotoxicity, NO production, and iNOS expression. The molecular mechanism by which SIRT1 inhibits iNOS expression appeared to involve the inhibition of the NF-kappaB signaling pathway through deacetylation of p65. In addition, SIRT1 activation by either resveratrol or adenoviral-directed overexpression of SIRT1 could prevent cytokine toxicity and maintain normal insulin-secreting responses to glucose in isolated rat islets. CONCLUSIONS: This study will provide valuable information not only into the mechanisms underlying beta-cell destruction but also into the regulation of SIRT1 as a possible target to attenuate cytokine-induced beta-cell damage.

Troglitazone enhances tamoxifen-induced growth inhibitory activity of MCF-7 cells.

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been identified as a potential source of therapy for human cancers. However, PPARgamma ligands have a limitation for breast cancer therapy, since estrogen receptor alpha (ER(alpha)) negatively interferes with PPARgamma signaling in breast cancer cells. Here we show that ER(alpha) inhihits PPARgamma transactivity and ER(alpha)-mediated inhibition of PPARgamma transactivity is blocked by tamoxifen, an estrogen receptor blocker. The activation of ER(alpha) with 17-beta-estradiol blocked PPRE transactivity induced by troglitazone, a PPARgamma ligand, indicating the resistance of ER(alpha)-positive breast cancer cells to troglitazone. Indeed, troglitazone inhibited the growth of ER(alpha)-negative MDA-MB-231 cells more than that of ER(alpha)-positive MCF-7 cells. Combination of troglitazone with tamoxifen led to a marked increase in growth inhibition of ER(alpha)-positive MCF-7 cells compared to either agent alone. Our data indicates that troglitazone enhances the growth inhibitory activity of tamoxifen in ER(alpha)-positive MCF-7 cells.