A venetoclax and azacitidine bridge-to-transplant strategy for NPM1-mutated acute myeloid leukaemia in molecular failure.

NPM1-mutated acute myeloid leukaemia (NPM1mut AML) represents a mostly favourable/intermediate risk disease that benefits from allogeneic haematopoietic stem cell transplantation (HSCT) in case of measurable residual disease (MRD) relapse or persistence after induction chemotherapy. Although the negative prognostic role of pre-HSCT MRD is established, no recommendations are available for the management of peri-transplant molecular failure (MF). Based on the efficacy data of venetoclax (VEN)-based treatment in NPM1mut AML older patients, we retrospectively analysed the off-label combination of VEN plus azacitidine (AZA) as bridge-to-transplant strategy in 11 NPM1mut MRD-positive fit AML patients. Patients were in MRD-positive complete remission (CRMRDpos ) at the time of treatment: nine in molecular relapse and two in molecular persistence. After a median number of two cycles (range 1-4) of VEN-AZA, 9/11 (81.8%) achieved CRMRD -negative (CRMRDneg ). All 11 patients proceeded to HSCT. With a median follow-up from treatment start of 26 months, and a median post-HSCT follow-up of 19 months, 10/11 patients are alive (1 died from non-relapse mortality), and 9/10 patients are in MRDneg status. This patient series highlights the efficacy and safety of VEN-AZA to prevent overt relapse, achieve deep responses and preserve patient fitness before HSCT, in patients with NPM1mut AML in MF.

Simulating the selfing and migration of Luehea divaricata populations in the Pampa biome to investigate the conservation potential of their genetic resources.

Computer simulations are an important tool for developing conservation strategies for forest species. This study used simulations to investigate the genetic, ecological, and reproductive patterns that contribute to the genetic structure of the tree Luehea divaricata Mart. & Zucc. in five forest fragments in the Brazilian Pampa biome. Using the EASYPOP model, we determined the selfing and migration rates that would match the corresponding genetic structure of microsatellite marker data (based on observed and expected heterozygosity parameters). The simulated reproductive mode was mixed, with a high rate of outcrossing (rate = 0.7). This was consistent with a selfing-incompatible system in this species, which reduced, but did not prevent, selfing. The simulated migration rate was 0.02, which implied that the forest fragments were isolated by distance, and that the inbreeding coefficients were high. Based on Nei's gene diversity analysis, 94% of the genetic variability was distributed within the forest fragments, and only 6% of the genetic diversity was caused by differences between them. Furthermore, the minimum viable population and minimum viable area genetic conservation parameters (which determine conservation potential in the short and long term) suggested that only the Inhatinhum forest fragment had the short-term potential to maintain its genetic diversity. However, in the long term, none of the forest fragments proved to be sustainable, indicating that the populations will require intervention to prevent a decline in genetic variability. The creation of ecological corridors could be a useful solution to connect forest fragments and enhance gene flow between them.

Advances in clinical NK cell studies: Donor selection, manufacturing and quality control.

Natural killer (NK) cells are increasingly used in clinical studies in order to treat patients with various malignancies. The following review summarizes platform lectures and 2013-2015 consortium meetings on manufacturing and clinical use of NK cells in Europe and United States. A broad overview of recent pre-clinical and clinical results in NK cell therapies is provided based on unstimulated, cytokine-activated, as well as genetically engineered NK cells using chimeric antigen receptors (CAR). Differences in donor selection, manufacturing and quality control of NK cells for cancer immunotherapies are described and basic recommendations are outlined for harmonization in future NK cell studies.

Multiplicação in vitro de segmentos apicais caulinares de segurelha (Satureja hortensis L. ) / In vitro multiplication of apical shoot segments of Summer Savory (Satureja hortensis L. )

O presente estudo teve por objetivo avaliar o efeito de BAP, na presença e ausência de ANA, sobre a multiplicação in vitro de segmentos apicais caulinares de Satureja hortensis. Os explantes foram isolados de plântulas germinadas in vitro e cultivados em meio nutritivo MS. O delineamento experimental utilizado foi o inteiramente casualizado, em esquema fatorial 2x5, correspondendo às concentrações de ANA (0 e 1 µM) e de BAP (0; 5; 10; 15 e 20 µM), com seis repetições, cada uma composta por três explantes. Para a porcentagem de explantes com brotações houve efeito significativo para o fator BAP, aumentando à medida que cresce a concentração da citocinina. A variável "número de brotos por explante" apresentou interação entre os fatores, havendo a maior formação de brotos na presença de ANA na faixa de 10 - 15 µM de BAP. Na presença da auxina, o maior valor ocorreu com 15 µM de BAP tendendo a diminuir independente da presença de ANA. O enraizamento dos segmentos apicais foi maior na presença de ANA e ausência de BAP, diminuindo com o aumento da citocinina. O número de folhas foi influenciado pela maior concentração de BAP sendo que a 15 µM foi observado o maior número, tendendo à queda com aumento da concentração. A concentração de 15 µM de BAP, independente de ANA, proporcionou os melhores resultados na multiplicação da espécie produzindo aumento de brotações e número de folhas, à exceção do enraizamento que foi influenciado pela auxina.

This study aimed to evaluate the effect of BAP in the presence and absence of NAA, on the in vitro multiplication of shoot apical segments of Satureja hortensis. The explants were isolated from seedlings germinated in vitro and cultured in nutrient medium MS. The experimental design was completely randomized in a 2x5 factorial arrangement, corresponding to the concentrations of NAA (0 and 1 µM) and BAP (0, 5, 10, 15 and 20 µM), with six replicates, each consisting of three explants. For the percentage of explants with shoots, there was no significant effect for the factor BAP, increasing as the concentration of BAP grows. The variable number of shoots per explant showed interaction between factors, with the highest shoot formation in the presence of NAA and BAP in the range of 10 -15 µM. In the presence of auxin, the highest value occurred with 15 µM BAP, tending to decline regardless of the presence of NAA. The rooting of the apical segments was higher in the presence of NAA and absence of BAP, decreasing with increasing cytokinin. Leaf number was influenced by the higher concentration of BAP, and the amount of 15 µMhadthe largest number, tending to decrease with increasing concentration. The concentration of 15 µM BAP, regardless of NAA, provided better results in the multiplication of the species, producing increased number of shoots and leaves, except for rooting, whichwas influenced by auxin.

Vaginal delivery rate in post-term pregnancies with one versus more than one dinoprostone gel administrations: an observational study.

AIM: The study aims to calculate the probability of a successful vaginal delivery in post-term low-risk women by using a set of predictors (maternal, fetal, and ultrasonographic) according to the number of dinoprostone gel applications. METHODS: This was an observational study of a cohort of 174 low-risk post-term singleton pregnancies. Parity, cervical status and length, and amniotic fluid volume (AFI) were evaluated immediately before prostaglandin gel induction at the Department of Obstetrics and Gynecology, University of Bologna, Bologna, Italy between January 2010 and October 2011. RESULTS: A consistent difference in vaginal delivery rates was observed for women who had one gel administration (Group 1) versus those who received either two or three gel administrations (Group 2): 77.5% at 24 hours and 97.4% at 48 hours vs. 27% at 24 hours and 54% at 48 hours (P <0.001 for both the comparisons) respectively. The predictors of a vaginal delivery were cervix dilatation and short cervix for Group 1, lower AFI for Group 2, and parity for both groups. CONCLUSION: Women who require more than one gel administration have a lower rate of vaginal delivery at 24 and 48 hours. Maternal, fetal, and ultrasonographic parameters can predict a successful vaginal delivery.

Outcome of severe placental insufficiency with abnormal umbilical artery Doppler prior to fetal viability.

OBJECTIVE: To evaluate the outcome of pregnancies complicated by placental insufficiency and abnormal umbilical artery Doppler prior to viability. DESIGN: A retrospective cohort study. SETTING: Italy. POPULATION: Singleton pregnancies with fetal growth restriction and absence of end-diastolic velocities (AEDVs) in the umbilical arteries prior to 24 weeks. METHODS: A retrospective cohort study of singleton pregnancies with fetal growth restriction and AEDVs in the umbilical arteries prior to 24 weeks. MAIN OUTCOME MEASURES: Fetal growth restriction and AEDVs in the umbilical arteries prior to 24 weeks. RESULTS: Of 16 fetuses first seen at 20-23 weeks, only 12 survived and one of these developed cerebral palsy. Severe hypertensive disorders occurred in three mothers. In four women, the Doppler waveforms progressively improved and developed a normal pulsatility. These fetuses had a better outcome than those that had persistent alterations: they were delivered later (34 versus 28 weeks), had a larger birthweight (1598 versus 630 g) and developed fewer complications. CONCLUSIONS: Placental insufficiency with AEDV in the umbilical arteries prior to fetal viability is associated with a high probability of perinatal death and neonatal complications. However, progressive amelioration of Doppler indices occurs in a subset of women, and these fetuses have a much better outcome.

Occurrence of septic shock in a patient submitted to emergency cerclage following a negative amniocentesis: report of a case.

BACKGROUND: Second trimester emergency cerclage is an option for pregnant women presenting bulging fetal membranes. Despite a significant prolongation of pregnancy might be achieved, serious fetal and maternal events have been reported. Exclusion of infections through preprocedure amniocentesis has been proposed. METHODS: A 37-year-old woman, gravida 4 para 1, was admitted at 21 weeks of gestation to our University Hospital due to bulging fetal membranes. An amniocentesis was performed in order to exclude an actual amniotic infection. Our Microbiology Department found a negative amniotic culture for bacteria and Mycoplasma and a normal glucose and interleukin-6 level, so a cervical cerclage was performed. The patient was discharged home on oral erythromycin. RESULTS: After 48 h, the patient complained of hyperpyrexia, shivers and reduced fetal movements. Ultrasound at admission showed absent cardiac activity and after cerclage removal a non-viable fetus was delivered vaginally. Piperacillin and tazobactam were started, but the clinical course of the patient deteriorated and she developed a cold septic shock and was submitted to hysterectomy and transferred to the ICU of our hospital. CONCLUSION: This report heralds that even after negative amniocentesis, a life-threatening infection may not be excluded in women candidate for emergency cerclage due to bulging fetal membranes.

Induction of regulatory T Cells by dendritic cells through indoleamine 2,3-dioxygenase: a potent mechanism of acquired peripheral tolerance.

Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that catalyzes the initial rate-limiting step in tryptophan degradation along the kynurenine pathway. Recent works have demonstrated a crucial role for IDO in the induction of immune tolerance during infections, pregnancy, transplantation, autoimmunity, and neoplasias. IDO is widely expressed in human tissues and cell subsets, including dendritic cells, where it modulates their function by increasing tolerogenic capacities. The aim of the present paper is to highlight the most recent data about IDO expression in dendritic cells and its role as a potent inducer of T regulatory cells.

The addition of plerixafor is safe and allows adequate PBSC collection in multiple myeloma and lymphoma patients poor mobilizers after chemotherapy and G-CSF.

We report 13 multiple myeloma (MM) or lymphoma patients who were failing PBSC mobilization after disease-specific chemotherapy and granulocyte-CSF (G-CSF), and received plerixafor to successfully collect PBSCs. Patients were considered poor mobilizers when the concentration of PB CD34(+) cells was always lower than 10 cells/µL, during the recovery phase after chemotherapy and/or were predicted to have inadequate PBSC collection to proceed to autologous transplantation. Plerixafor (0.24 mg/kg) was administered subcutaneously for up to three consecutive days, while continuing G-CSF, 10-11 h before the planned leukapheresis. Plerixafor administration was safe and no significant adverse events were recorded. We observed a 4.7 median fold-increase in the number of circulating CD34(+) cells after plerixafor as compared with baseline CD34(+) cell concentration (from a median of 6.2 (range 1-12) to 21.5 (range 9-88) cells/µL). All patients collected >2 × 10(6) CD34(+) cells/kg in 1-3 leukaphereses. In all, 5/13 patients have already undergone autograft with plerixafor-mobilized PBSCs, showing a rapid and durable hematological recovery. Our results suggest that the pre-emptive addition of plerixafor to G-CSF after chemotherapy is safe and may allow the rescue of lymphoma and MM patients, who need autologous transplantation but are failing PBSC mobilization.

Metal-related artifacts in instrumented spine. Techniques for reducing artifacts in CT and MRI: state of the art.

The projectional nature of radiogram limits its amount of information about the instrumented spine. MRI and CT imaging can be more helpful, using cross-sectional view. However, the presence of metal-related artifacts at both conventional CT and MRI imaging can obscure relevant anatomy and disease. We reviewed the literature about overcoming artifacts from metallic orthopaedic implants at high-field strength MRI imaging and multi-detector CT. The evolution of multichannel CT has made available new techniques that can help minimizing the severe beam-hardening artifacts. The presence of artifacts at CT from metal hardware is related to image reconstruction algorithm (filter), tube current (in mA), X-ray kilovolt peak, pitch, hardware composition, geometry (shape), and location. MRI imaging has been used safely in patients with orthopaedic metallic implants because most of these implants do not have ferromagnetic properties and have been fixed into position. However, on MRI imaging metallic implants may produce geometric distortion, the so-called susceptibility artifact. In conclusion, although 140 kV and high milliamperage second exposures are recommended for imaging patients with hardware, caution should always be exercised, particularly in children, young adults, and patients undergoing multiple examinations. MRI artifacts can be minimized by positioning optimally and correctly the examined anatomy part with metallic implants in the magnet and by choosing fast spin-echo sequences, and in some cases also STIR sequences, with an anterior to posterior frequency-encoding direction and the smallest voxel size.

Abdominal ultrasonography in inheredited diseases of carbohydrate metabolism.

PURPOSE: To determine the usefulness of abdominal sonography in inherited diseases of carbohydrate metabolism. MATERIALS AND METHODS: Thirty patients (age range, 4 months to 27 years) with glycogen storage diseases, galactosemia, disorders of fructose metabolism were studied with sonography. Echogenicity of the liver, sonographic dimensions of liver, kidneys and spleen were evaluated. Plasma blood parameters (ALT, AST, total cholesterol, triglycerides) were determined. RESULTS: Liver was enlarged in 21/22 patients (95.4%) with glycogen storage diseases, in both subjects with disorders of fructose metabolism, and in 2/6 patients (33.3%) with galactosemia. Hepatic echogenicity was increased in 20/22 patients (90.9%) with glycogen storage diseases, and in the subject with hereditary fructose intolerance. Patients with galactosemia did not show increased liver echogenicity. Both kidneys were enlarged in 8/17 patients (47.0%) with glycogen storage disease type I. Subjects with increased hepatic echogenicity exhibited higher plasma concentrations of any blood parameter than the others with normal echogenicity (p<0.05). CONCLUSIONS: Sonography can be useful in identification of inherited diseases of carbohydrate metabolism even if further examinations are necessary for an ultimate diagnosis.

[Isolated muscular sarcoidosis mimicking a tumoral lesion]. / Manifestazione isolata di sarcoidosi muscolare simulante una lesione tumorale.

We report a case of a woman with a palpable painful nodule on her left leg. MR and CT showed a lesion that could be described as a neoplasm. Excisonal biopsy revealed a noncaseating granuloma. The woman presented the nodular type of muscular isolated sarcoidosis. Further the disease involved the lungs; this confirmed the accurate diagnosis of sarcoidosis. Sarcoidosis is a chronic, multisystem granulomatous disease of unknown etiology. Muscle involvement is frequent, but often asymptomatic. There are three forms of muscular sarcoidosis: only the nodular type can be recognized by technical imaging. MR and ultrasound are the best methods to attempt the diagnosis of nodular muscular sarcoidosis; nevertheless, the lesion must have a standardized behaviour because it can mimic a malignant neoform. In this case, biopsy is the only tool to identify the disease.

Immunotoxins containing recombinant anti-CTLA-4 single-chain fragment variable antibodies and saporin: in vitro results and in vivo effects in an acute rejection model.

Immunotoxins containing recombinant human-derived single-chain fragment variable (scFv) reagents (83 and 40) against CTLA-4 (CD152) linked to saporin, a ribosome-inactivating protein, were prepared and tested on CD3/CD28-activated T lymphocytes, MLRs, CTLA-4-positive cell lines, and hemopoietic precursors. Immunotoxins induced apoptosis in activated T lymphocytes and were able to specifically inhibit MLR between T lymphocytes and dendritic cells. The 83-saporin immunotoxin also inhibited the T cell activation in an MLR between T lymphocytes and an EBV-positive lymphoblastoid B cell line. Toxicity tests on hemopoietic precursors showed little or no effects in inhibiting colonies' growth. As the 83 scFv Ab was reactive also with activated mouse T lymphocytes, 83-saporin was tested in a model of tumor rejection consisting of C57BL/6 mice bearing a murine H.end endothelioma cell line, derived from DBA/2 mice. The lymphoid infiltration due to the presence of the tumor was reduced to a high extent, demonstrating that the immunotoxin was actually available and active in vivo. Thus, taking the results altogether, this study might represent a new breakthrough for immunotherapy, showing the possibility of targeting CTLA-4 to kill activated T cells, using conjugates containing scFv Abs and type 1 ribosome-inactivating protein.

Dendritic cell differentiation from hematopoietic CD34+ progenitor cells.

Dendritic cells (DC) are the most powerful antigen presenting cells (APC) and play a pivotal role in initiating the immune response. In light of their unique properties, DC have been proposed as a tool to enhance immunity against infectious agents and in anticancer vaccine strategies. In the last few years, the development of DC has been extensively investigated. The present paper summarizes the most recent findings on the differentiation of myeloid DC from hematopoietic CD34+ progenitors and methods for DC generation in vitro. A better understanding of DC function has important implications for their use in clinical settings.

Interleukin-11 induces Th2 polarization of human CD4(+) T cells.

Exploration of the immunomodulatory activities of the multifunctional cytokine interleukin-11 (IL-11) has prompted several therapeutic applications. The immunomodulatory effects of IL-11 on human antigen-presenting cells and on T cells were investigated. IL-11 inhibited IL-12 production by activated CD14(+) monocytes, but not by mature dendritic cells (DCs) stimulated via CD40 ligation. Moreover, IL-11 did not affect either DC maturation, as demonstrated by phenotypic analysis and evaluation of cytokine production, or DC generation from progenitor cells in the presence of specific growth factors. Molecular analysis demonstrated the expression of IL-11 receptor messenger RNA in highly purified CD14(+) monocytes, CD19(+) B cells, CD8(+), and CD4(+) T cells, and CD4(+)CD45RA(+) naive T lymphocytes. In keeping with this finding, IL-11 directly prevented Th1 polarization of highly purified CD4(+)CD45RA(+) naive T cells stimulated with anti-CD3/CD28 antibodies, as demonstrated by significant increases of IL-4 and IL-5, by significantly decreased interferon-gamma production and by flow cytometry intracellular staining of cytokines. Coincubation of naive T cells with DCs, the most potent stimulators of Th1 differentiation, did not revert IL-11-mediated Th2 polarization. Furthermore, parallel experiments demonstrated that the activity of IL-11 was comparable with that induced by IL-4, the most effective Th2-polarizing cytokine. Taken together, these findings show that IL-11 inhibits Th1 polarization by exerting a direct effect on human T lymphocytes and by reducing IL-12 production by macrophages. Conversely, IL-11 does not exert any activity on DCs. This suggests that IL-11 could have therapeutic potential for diseases where Th1 responses play a dominant pathogenic role.

Stem cell factor and FLT3-ligand are strictly required to sustain the long-term expansion of primitive CD34+DR- dendritic cell precursors.

We studied cytokine-driven differentiation of primitive human CD34(+)HLA-DR(-) cells to myeloid dendritic cells (DC). Hemopoietic cells were grown in long-term cultures in the presence of various combinations of early acting cytokines such as FLT3-ligand (FLT3-L) and stem cell factor (SCF) and the differentiating growth factors GM-CSF and TNF-alpha. Two weeks of incubation with GM-CSF and TNF-alpha generated fully functional DC. However, clonogenic assays demonstrated that CFU-DC did not survive beyond 1 wk in liquid culture regardless of whether FLT3-L and/or SCF were added. FLT3-L or SCF alone did not support DC maturation. However, the combination of the two early acting cytokines allowed a 100-fold expansion of CFU-DC for >1 month. Phenotypic analysis demonstrated the differentiation of CD34(+)DR(-) cells into CD34(-)CD33(+)DR(+)CD14(+) cells, which were intermediate progenitors capable of differentiating into functionally active DC upon further incubation with GM-CSF and TNF-alpha. As expected, GM-CSF and TNF-alpha generated DC from committed CD34(+)DR(+) cells. However, only SCF, with or without FLT3-L, induced the expansion of DC precursors for >4 wk, as documented by secondary clonogenic assays. This demonstrates that although GM-CSF and TNF-alpha do not require additional cytokines to generate DC from primitive human CD34(+)DR(-) progenitor cells, they do force terminal differentiation of DC precursors. Conversely, FLT3-L and SCF do not directly affect DC differentiation, but instead sustain the long-term expansion of CFU-DC, which can be induced to produce mature DC by GM-CSF and TNF-alpha.