MagRET Nanoparticles: An Iron Oxide Nanocomposite Platform for Gene Silencing from MicroRNAs to Long Noncoding RNAs.

Silencing of RNA to knock down genes is currently one of the top priorities in gene therapies for cancer. However, to become practical the obstacle of RNA delivery needs to be solved. In this study, we used innovative maghemite (γ-Fe2O3) nanoparticles, termed magnetic reagent for efficient transfection (MagRET), which are composed of a maghemite core that is surface-doped by lanthanide Ce(3/4+) cations using sonochemistry. Thereafter, a polycationic polyethylenimine (PEI) polymer phase is bound to the maghemite core via coordinative chemistry enabled by the [CeL(n)](3/4+)cations/complex. PEI oxidation was used to mitigate the in vivo toxicity. Using this approach, silencing of 80-100% was observed for mRNAs, microRNAs, and lncRNA in a variety of cancer cells. MagRET NPs are advantageous in hard to transfect leukemias. This versatile nanoscale carrier can silence all known types of RNAs and these MagRET NPs with oxidized PEI are not lethal upon injection, thus holding promise for therapeutic applications, as a theranostic tool.

The KMT2A recombinome of acute leukemias in 2023.

Chromosomal rearrangements of the human KMT2A/MLL gene are associated with de novo as well as therapy-induced infant, pediatric, and adult acute leukemias. Here, we present the data obtained from 3401 acute leukemia patients that have been analyzed between 2003 and 2022. Genomic breakpoints within the KMT2A gene and the involved translocation partner genes (TPGs) and KMT2A-partial tandem duplications (PTDs) were determined. Including the published data from the literature, a total of 107 in-frame KMT2A gene fusions have been identified so far. Further 16 rearrangements were out-of-frame fusions, 18 patients had no partner gene fused to 5'-KMT2A, two patients had a 5'-KMT2A deletion, and one ETV6::RUNX1 patient had an KMT2A insertion at the breakpoint. The seven most frequent TPGs and PTDs account for more than 90% of all recombinations of the KMT2A, 37 occur recurrently and 63 were identified so far only once. This study provides a comprehensive analysis of the KMT2A recombinome in acute leukemia patients. Besides the scientific gain of information, genomic breakpoint sequences of these patients were used to monitor minimal residual disease (MRD). Thus, this work may be directly translated from the bench to the bedside of patients and meet the clinical needs to improve patient survival.

Trp207Gly in platelet glycoprotein Ibalpha is a novel mutation that disrupts the connection between the leucine-rich repeat domain and the disulfide loop structure and causes Bernard-Soulier syndrome.

BACKGROUND: Bernard-Soulier syndrome (BSS) is a severe inherited bleeding disorder that is caused by a defect in glycoprotein (GP)Ib-IX-V complex, the platelet membrane receptor for von Willebrand factor. PATIENTS: The diagnosis of BSS was made in two members of a Bukharian Jewish family who had life-long thrombocytopenia associated with mucocutaneous bleeding manifestations. METHODS AND RESULTS: Flow cytometry and Western blot analyses showed only trace amounts of GPIb and GPIX on the patients' platelets. Sequence analysis of the GPIbalpha gene revealed a homozygous T > G transversion at nucleotide 709 predicting Trp207Gly substitution in the mature protein. Introduction of the mutation into a mammalian expression construct abolished the surface expression of GPIbalpha in transfected baby hamster kidney cells. The crystal structure of the N-terminus of GPIbalpha (PDB: 1SQ0) indicates that Trp207 is completely buried and located in a disulfide loop structure that interacts with the leucine-rich repeat (LRR) domain. CONCLUSION: A novel mutation, Trp207Gly, causes BSS and predicts disruption of the interaction between a disulfide loop and the LRR domain that is essential for the integrity of GPIbalpha structure.

Chromosomal aberrations and gene expression profiles in non-small cell lung cancer.

Alterations in genomic content and changes in gene expression levels are central characteristics of tumors and pivotal to the tumorigenic process. We analyzed 23 non-small cell lung cancer (NSCLC) tumors by array comparative genomic hybridization (array CGH). Aberrant regions identified included well-characterized chromosomal aberrations such as amplifications of 3q and 8q and deletions of 3p21.31. Less frequently identified aberrations such as amplifications of 7q22.3-31.31 and 12p11.23-13.2, and previously unidentified aberrations such as deletion of 11q12.3-13.3 were also detected. To enhance our ability to identify key acting genes residing in these regions, we combined array CGH results with gene expression profiling performed on the same tumor samples. We identified a set of genes with concordant changes in DNA copy number and expression levels, i.e. overexpressed genes located in amplified regions and underexpressed genes located in deleted regions. This set included members of the Wnt/beta-catenin pathway, genes involved in DNA replication, and matrix metalloproteases (MMPs). Functional enrichment analysis of the genes both overexpressed and amplified revealed a significant enrichment for DNA replication and repair, and extracellular matrix component gene ontology annotations. We verified the changes in expressions of MCM2, MCM6, RUVBL1, MMP1, MMP12 by real-time quantitative PCR. Our results provide a high resolution map of copy number changes in non-small cell lung cancer. The joint analysis of array CGH and gene expression analysis highlights genes with concordant changes in expression and copy number that may be critical to lung cancer development and progression.

Utilizing somatic mutation data from numerous studies for cancer research: proof of concept and applications.

Large cancer projects measure somatic mutations in thousands of samples, gradually assembling a catalog of recurring mutations in cancer. Many methods analyze these data jointly with auxiliary information with the aim of identifying subtype-specific results. Here, we show that somatic gene mutations alone can reliably and specifically predict cancer subtypes. Interpretation of the classifiers provides useful insights for several biomedical applications. We analyze the COSMIC database, which collects somatic mutations from The Cancer Genome Atlas (TCGA) as well as from many smaller scale studies. We use multi-label classification techniques and the Disease Ontology hierarchy in order to identify cancer subtype-specific biomarkers. Cancer subtype classifiers based on TCGA and the smaller studies have comparable performance, and the smaller studies add a substantial value in terms of validation, coverage of additional subtypes, and improved classification. The gene sets of the classifiers are used for threefold contribution. First, we refine the associations of genes to cancer subtypes and identify novel compelling candidate driver genes. Second, using our classifiers we successfully predict the primary site of metastatic samples. Third, we provide novel hypotheses regarding detection of subtype-specific synthetic lethality interactions. From the cancer research community perspective, our results suggest that curation efforts, such as COSMIC, have great added and complementary value even in the era of large international cancer projects.

The histone deacetylase inhibitor givinostat (ITF2357) exhibits potent anti-tumor activity against CRLF2-rearranged BCP-ALL.

Leukemias bearing CRLF2 and JAK2 gene alterations are characterized by aberrant JAK/STAT signaling and poor prognosis. The HDAC inhibitor givinostat/ITF2357 has been shown to exert anti-neoplastic activity against both systemic juvenile idiopathic arthritis and myeloproliferative neoplasms through inhibition of the JAK/STAT pathway. These findings led us to hypothesize that givinostat might also act against CRLF2-rearranged BCP-ALL, which lack effective therapies. Here, we found that givinostat inhibited proliferation and induced apoptosis of BCP-ALL CRLF2-rearranged cell lines, positive for exon 16 JAK2 mutations. Likewise, givinostat killed primary cells, but not their normal hematopoietic counterparts, from patients carrying CRLF2 rearrangements. At low doses, givinostat downregulated the expression of genes belonging to the JAK/STAT pathway and inhibited STAT5 phosphorylation. In vivo, givinostat significantly reduced engraftment of human blasts in patient-derived xenograft models of CRLF2-positive BCP-ALL. Importantly, givinostat killed ruxolitinib-resistant cells and potentiated the effect of current chemotherapy. Thus, givinostat in combination with conventional chemotherapy may represent an effective therapeutic option for these difficult-to-treat subsets of ALL. Lastly, the selective killing of cancer cells by givinostat may allow the design of reduced intensity regimens in CRLF2-rearranged Down syndrome-associated BCP-ALL patients with an overall benefit in terms of both toxicity and related complications.

The biology, pathogenesis and clinical aspects of acute lymphoblastic leukemia in children with Down syndrome.

Children with Down syndrome (DS) are at a 20-fold increased risk for acute lymphoblastic leukemia (DS-ALL). Although the etiology of this higher risk of developing leukemia remains largely unclear, the recent identification of CRLF2 (cytokine receptor like factor 2) and JAK2 mutations and study of the effect of trisomy of Hmgn1 and Dyrk1a (dual-specificity tyrosine phosphorylation-regulated kinase 1A) on B-cell development have shed significant new light on the disease process. Here we focus on the clinical features, biology and genetics of ALL in children with DS. We review the unique characteristics of DS-ALL on both the clinical and molecular levels and discuss the differences in treatments and outcomes in ALL in children with DS compared with those without DS. The identification of new biological insights is expected to pave the way for novel targeted therapies.

Monitoring of residual disease in chronic myelogenous leukemia by quantitative polymerase chain reaction.

The polymerase chain reaction (PCR) has become a standard method for highly sensitive detection of the bcr/abl rearrangement in patients with chronic myelogenous leukemia (CML) or acute lymphoblastic leukemia (ALL). The exquisite sensitivity of the PCR facilitates the detection of residual leukemic cells after chemotherapy or after bone marrow transplantation. However, the detection of minimal residual disease does not yield any information on the malignant potential of the bcr/abl-rearranged cells. Qualitative PCR is therefore of limited prognostic value in the monitoring of residual leukemia. We have adapted the PCR for quantitative evaluation of cells carrying the bcr/abl rearrangement and by means of two exemplary cases of CML patients after bone marrow transplantation and under treatment with alpha-interferon, respectively, we show that this new technique is suitable for the long term follow-up of the activity of the residual bcr/abl rearranged clone. Longitudinal monitoring of residual disease by the technique presented provides a novel tool for detection of incipient relapse at a very early stage.

Expression of identical E2A/PBX1 fusion transcripts occurs in both pre-B and early pre-B immunological subtypes of childhood acute lymphoblastic leukemia.

The translocation t(1;19)(q23;p13) in pediatric patients with acute lymphoblastic leukemia (ALL) was demonstrated in early reports to result in a consistent fusion between the E2A and PBX1 gene sequences and in the expression of a uniform, chimeric E2A/PBX1 mRNA with transforming potential. More recent observations suggested that cytogenetically identical t(1;19) translocations can result in the transcription of different mRNA species and that expression of the E2A/PBX1 message may be restricted to patients with the t(1;19) who display a pre-B phenotype of ALL. To further assess the correlation between the immunologic subtypes of the disease and specific genetic alterations, we have performed cytogenetic and molecular analyses in 221 children with B-lineage ALL. Expression of the chimeric E2A/PBX1 message was detected in 21 patients. Out of 14 patients, in whom cytoplasmic immunoglobulin (cig) analyses were available, no less than four had a cig- B-cell precursor ALL, whereas 10 displayed a cig+ B-ALL immunophenotype. These findings are at variance with a recent report in which expression of the E2A/PBX1 message was linked exclusively to a subset of patients who displayed a cig+ pre-B-cell precursor phenotype of ALL. In seven cases diagnosed before 1986, cig analyses were not available, and consequently E2A/PBX1 expression could not be correlated to a particular immunological subtype of B-cell precursor ALL. Our results have important implications for the detection of residual disease in pediatric patients where expression of the typical E2A/PBX1 mRNA may occur both in cig+ (pre-B) and cig- (early pre-B) immunologic subtypes of ALL.

Detection and clinical relevance of genetic abnormalities in pediatric acute lymphoblastic leukemia: a comparison between cytogenetic and polymerase chain reaction analyses.

The E2A/PBX1 and the BCR/ABL fusion genes result from the t(1;19)(q23;p13) and the t(9;22)(q34;q11), respectively, and encode oncoproteins which are thought to play an important role in the development of acute lymphoblastic leukemia (ALL) subtypes associated with adverse prognosis. The use of the polymerase chain reaction (PCR) for the detection of these genetic rearrangements may offer advantages over cytogenetic techniques which are often unsatisfactory in patients with ALL and, furthermore, provide a useful tool for monitoring of residual disease. However, it has not yet been evaluated whether the employment of PCR at the time of diagnosis improves the detection rate of these clinically relevant genetic anomalies. We have developed a multiprimer-PCR protocol which facilitates the detection of each of the four chimeric E2A/PBX1 and BCR/ABL mRNAs in a single reaction. This protocol was used for the evaluation of bone-marrow or blood samples from 251 children with ALL in whom cytogenetic analyses had been performed. Of the 251 patients, 221 had a B-cell precursor immunophenotype. In this group, 21 patients (9.5%) carrying the E2A/PBX1 rearrangement and three patients (1.4%) with BCR/ABL transcripts were detected by PCR. Twelve of these cases had escaped the detection by conventional cytogenetic analysis. In two of 12 patients with a typical t(1;19)(q23;p13), no E2A/PBX1 transcripts were identified by PCR, thus suggesting the presence of different molecular rearrangements. Residual leukemic cells were detected by PCR in five of eight patients who were followed during complete clinical remission. The frontline use of PCR has an important impact on the timely diagnosis, therapeutic decisions, and monitoring of high-risk patients with B-cell precursor leukemia who carry the E2A/PBX1 or BCR/ABL fusion genes.

Overexpression of ERG in cord blood progenitors promotes expansion and recapitulates molecular signatures of high ERG leukemias.

High expression of the ETS family transcription factor ERG is associated with poor clinical outcome in acute myeloid leukemia (AML) and acute T-cell lymphoblastic leukemia (T-ALL). In murine models, high ERG expression induces both T-ALL and AML. However, no study to date has defined the effect of high ERG expression on primary human hematopoietic cells. In the present study, human CD34+ cells were transduced with retroviral vectors to elevate ERG gene expression to levels detected in high ERG AML. RNA sequencing was performed on purified populations of transduced cells to define the effects of high ERG on gene expression in human CD34+ cells. Integration of the genome-wide expression data with other data sets revealed that high ERG drives an expression signature that shares features of normal hematopoietic stem cells, high ERG AMLs, early T-cell precursor-ALLs and leukemic stem cell signatures associated with poor clinical outcome. Functional assays linked this gene expression profile to enhanced progenitor cell expansion. These results support a model whereby a stem cell gene expression network driven by high ERG in human cells enhances the expansion of the progenitor pool, providing opportunity for the acquisition and propagation of mutations and the development of leukemia.

Pharmacological modulation of vagal cardiac control measured by heart rate power spectrum: a possible bioequivalent probe.

The autonomic cardiac control was studied as a sensitive parameter of anticholinergic treatment in humans, using heart-rate (HR) power spectrum. A cross-over placebo controlled study was performed in 8 young volunteers who received increasing bolusdoses of IV atropine (from 1.3 micrograms/kg to 29.9 micrograms/kg) or placebo. Computing the HR power spectrum and integrating over specific frequency bands, we focused in particular on the respiratory frequency band (usually between 0.2-0.4 Hz) which is purely of vagal mediation. At small atropine doses (less than 5.2 micrograms/kg), the respiratory peak increased, relative to baseline, with maximal response at 2.6 micrograms/kg (from 1.0 to 1.9 +/- 0.9). Larger doses of atropine (greater than or equal to 6.5 micrograms/kg) reduced the power of the respiratory peak, by a few orders of magnitude, in a dose-dependent way. Corresponding changes were observed in mean HR but in the opposite direction i.e., a maximal bradycardia at 2.6 micrograms/kg and a nearly two fold increase in mean HR at 29.9 micrograms/kg. We conclude that atropine has a bimodal dose-dependent effect on parasympathetic cardiac control. Since the use of HR spectral analysis has been demonstrated in various animal species, we suggest that it can be used as a sensitive noninvasive probe for animal to man transformation studies.

Paradoxical pharmacodynamic effect of atropine on parasympathetic control: a study by spectral analysis of heart rate fluctuations.

The power spectrum of instantaneous heart rate fluctuations was used to determine the optimal doses of atropine that induce a maximal vagolytic or vagomimetic effect. In a crossover placebo controlled study, eight volunteers received increasing bolus doses of intravenous atropine (0.1 to 2.3 mg per subject) or placebo, and frequency bands of the power spectrum were integrated. During atropine administration a significant bimodal dose dependence was observed for the respiratory peak (0.2 to 0.4 Hz, p = 0.0006), the midfrequency band (0.09 to 0.15 Hz, p = 0.0035), and mean heart rate (p < 0.0001). Low doses (< 0.4 mg per subject) increased the respiratory and midfrequency band power, with maximal response at 0.2 mg per subject. Larger doses of atropine, 0.5 to 2.3 mg per subject, markedly reduced the power in all frequency bands in a dose-dependent way. The corresponding changes in mean heart rate were simultaneous, but in the opposite direction. We suggest that the respiratory peak of the power spectrum can be used to optimize drug effects on cardiac parasympathetic control.

Albright hereditary osteodystrophy with hypothyroidism, normocalcemia, and normal Gs protein activity: a family presenting with congenital osteoma cutis.

The syndrome of Albright hereditary osteodystrophy (AHO), pseudohypoparathyroidism (PHP) and pseudopseudohypoparathyroidism (PPHP) is clinically and genetically heterogeneous. Classically, patients with PHP have the skeletal features of AHO, resistance to multiple hormones that work via cAMP such as parathyroid hormone and thyroid stimulating hormone, and deficient activity of Gs protein, the guanine nucleotide-binding protein that stimulates adenylate cyclase. However, patients without hormone resistance but with AHO and Gs deficiency were described (PPHP), as well as patients with multiple hormone resistance but without AHO or Gs deficiency. In a few patients with deficient Gs activity, hypothyroidism rather than hypocalcemia was the initial presentation of the disorder. We describe here a new variant of the syndrome, affecting 5 individuals in a 3 generation family with AHO, normal Gs activity and hypothyroidism. In the first 2 generations, mild features of AHO were present. The 2 sibs in the third generation had severe manifestations of AHO, including mild mental retardation as well as hypothyroidism. Diagnosis of congenital osteoma cutis at birth of the proband led to the diagnosis of the family. Elucidation of the molecular defect will shed light on the relationship between hormone resistance and AHO, as well as on the physiological mechanism of hormonal signal transduction.

Role of tissue diagnosis in pulmonary involvement in pediatric human immunodeficiency virus infection.

BACKGROUND: Pulmonary complications occur commonly during HIV infection. The aim of this study was to evaluate the clinical value of lung tissue examination in the diagnosis and treatment of pulmonary disorders in children with HIV infection. METHODS: The medical records of 347 children enrolled between January, 1990, and April, 1994, into various antiretroviral therapy protocols were reviewed to identify patients who underwent a lung biopsy. RESULTS: Fourteen patients underwent diagnostic lung biopsies on 16 separate occasions. The most common radiologic findings were nodular infiltrates which were localized in 7 patients and diffuse in 6. Eight patients presented with fever and progressive respiratory distress unresponsive to empiric therapy, whereas the rest had progressive nodular infiltrates. The pathologic diagnoses included opportunistic infection in 7 patients, lymphocytic interstitial pneumonitis in 5, non-Hodgkin's lymphoma in 3 and interstitial fibrosis in 1. The biopsy led to a major change in the treatment of 7 patients which resulted in a significant improvement of the pulmonary process in all of them. In an additional patient the excisional biopsy proved curative. CONCLUSIONS: When patients are selected appropriately, lung biopsy might have a significant impact on therapy and outcome in HIV-infected children with pulmonary infiltrates.

Tetraploid myeloid cells in donors of peripheral blood stem cells treated with rhG-CSF.

Recombinant human granulocyte colony-stimulating factor (rhG-CSF) is frequently used to mobilize CD34+ cells in healthy donors and patient with malignant diseases prior to peripheral blood stem cell (PBSC) harvest. To analyze the effects of rhG-CSF on morphology and genotype of white blood cells, a novel multiparametric cell scanning system that combines morphologic, immune and genotypic analyses of the same cells was used. We report here that tetraploid myeloid cells are present in the peripheral blood of donors treated with rhG-CSF. The tetraploidy was detected in up to 0.6% of differentiated myeloid cells and all observed CD34+ cells were diploid. Thus, short treatment with rhG-CSF of PBSC donors induces numerfical chromosomal alterations in a small subset of mature myeloid cells.

Modulation of the dose-dependent effects of atropine by low-dose pyridostigmine: quantification by spectral analysis of heart rate fluctuations in healthy human beings.

The interaction between a low-dose cholinesterase inhibitor, pyridostigmine (PYR), and atropine was investigated by spectral analysis of heart rate fluctuations in eight healthy humans. Each subject was given increasing boluses of IV atropine during treatment with PYR (30 mg.3/day) or placebo. PYR attenuated the bimodal dose-dependent changes in the respiratory peak (which respresents the parasympathetic control) in response to atropine. We suggest that spectral analysis can be used for quantifying the complex dose-dependent cholinergic agonist-antagonist interactions, and may help to disclose an asymptomatic low-dose intoxication with acetylcholinesterase inhibitors.

Determination of the "time of useful consciousness" (TUC) in repeated exposures to simulated altitude of 25,000 ft (7,620 m).

Time of useful consciousness (TUC) was determined in 17 subjects exposed twice to 25,000 ft (7,620 m) in an altitude chamber. The criterion for TUC determination was inability to add two-digit numbers correctly. Median values of TUC were 267.5 seconds (s) in the first exposure and 240 s in the second. The intraindividual variability between the two exposures was 40.6 s. The probability of remaining in "useful" consciousness as a function of time at 25,000 ft (7,620 m) was similar in both exposures. The need for a more scientific approach towards the determination of time of useful consciousness in simulated high altitudes is raised.

Ocular effects of hyoscine in double dose transdermal administration and its reversal by low dose pyridostigmine.

The potential of low dose (30 mg t.i.d) pyridostigmine to reduce the ocular side effects of double dose transdermal controlled release hyoscine was evaluated by the study of near visual acuity, accommodation amplitude and pupil diameter in a placebo controlled, double masked study. We studied 47 healthy men (age 18-21 yr) in 3 groups: 16 assigned to placebo hyoscine and placebo pyridostigmine, 15 assigned to double dose hyoscine and placebo pyridostigmine, and 16 to double dose hyoscine and pyridostigmine. Subjects were tested during 48 h of treatment and 48 h of washout period. Blood cholinesterase inhibition level and amount of hyoscine released from the patches were used as parameters of reliability. Difference between groups was assessed using change from baseline scores. Double dose hyoscine caused decrease in near visual acuity to a mean of 14/18. Accommodation amplitude was decreased in the double dose transdermal hyoscine group from 9.19 +/- 1.04 to 4.83 +/- 1.97 diopters of accommodation. This decrease was significant when compared to the placebo group (p < 0.05) and to the pyridostigmine-protected group (p < 0.05). Pyridostigmine, however, did not significantly change the hyoscine-induced mydriasis of 1.47 + 0.15 mm change from baseline (p < 0.05). These results suggest that pyridostigmine administration may be beneficial in shortening recovery time when near vision impairment is experienced following single and double dose transdermal hyoscine administration.

The effect of repeated doses of 30 mg pyridostigmine bromide on pilot performance in an A-4 flight simulator.

The effect of repeated doses of 30 mg pyridostigmine bromide every 8 h on flight skills in an A-4 simulator was tested in this crossover double-blind placebo-controlled study on 10 pilots experienced in actual and simulated A-4 flights. The pilots flew two test simulator flights 2 h after the fourth dose of pyridostigmine or placebo. The flight profile included navigation, rapid ascent, 360 degrees turns, and instrument landing. Each flight lasted approximately 20 min. Flight parameters measured included indicated air speed, true heading, barometric altitude, vertical velocity, and bank. The mean whole blood cholinesterase inhibition level was 29%. There was no decrement in performance under treatment with pyridostigmine in the percent of deviation time from the prescribed limits or in the average duration or magnitude of the deviation in each of the flight parameters. We conclude that pyridostigmine bromide in repeated doses of 30 mg every 8 h does not appear to influence pilot performance during short A-4 missions.