Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma

Background: Neuroblastoma is a childhood cancer derived from immature cells of the sympathetic nervous system. The disease is clinically heterogeneous, ranging from neuronal differentiated benign ganglioneuromas to aggressive metastatic tumours with poor prognosis. Amplification of the MYCN oncogene is a well established poor prognostic factor found in up to 40% of high risk neuroblastomas. Using neuroblastoma cell lines to study neuronal differentiation in vitro is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers. Results: In this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down MYCN expression in MYCN-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers NFL and GAP43. In addition, we show that induced expression of retrovirally delivered anti- MYCN shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced. Conclusion: We have developed an efficient MYCN-knockdown in vitro model system to study neuronal differentiation in MNA neuroblastomas

Conditional expression of retrovirally delivered anti-MYCN shRNA as an in vitro model system to study neuronal differentiation in MYCN-amplified neuroblastoma

<p>Abstract</p> <p>Background</p> <p>Neuroblastoma is a childhood cancer derived from immature cells of the sympathetic nervous system. The disease is clinically heterogeneous, ranging from neuronal differentiated benign ganglioneuromas to aggressive metastatic tumours with poor prognosis. Amplification of the MYCN oncogene is a well established poor prognostic factor found in up to 40% of high risk neuroblastomas.</p> <p>Using neuroblastoma cell lines to study neuronal differentiation <it>in vitro </it>is now well established. Several protocols, including exposure to various agents and growth factors, will differentiate neuroblastoma cell lines into neuron-like cells. These cells are characterized by a neuronal morphology with long extensively branched neurites and expression of several neurospecific markers.</p> <p>Results</p> <p>In this study we use retrovirally delivered inducible short-hairpin RNA (shRNA) modules to knock down <it>MYCN </it>expression in <it>MYCN</it>-amplified (MNA) neuroblastoma cell lines. By addition of the inducer doxycycline, we show that the Kelly and SK-N-BE(2) neuroblastoma cell lines efficiently differentiate into neuron-like cells with an extensive network of neurites. These cells are further characterized by increased expression of the neuronal differentiation markers <it>NFL </it>and <it>GAP43</it>. In addition, we show that induced expression of retrovirally delivered anti-<it>MYCN </it>shRNA inhibits cell proliferation by increasing the fraction of MNA neuroblastoma cells in the G1 phase of the cell cycle and that the clonogenic growth potential of these cells was also dramatically reduced.</p> <p>Conclusion</p> <p>We have developed an efficient <it>MYCN</it>-knockdown <it>in vitro </it>model system to study neuronal differentiation in MNA neuroblastomas.</p

DNA methylation holds prognostic information in relapsed precursor B-cell acute lymphoblastic leukemia

Background: Few biological markers are associated with survival after relapse of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In pediatric T-cell ALL, we have identified promoter-associated methylation alterations that correlate with prognosis. Here, the prognostic relevance of CpG island methylation phenotype (CIMP) classification was investigated in pediatric BCP-ALL patients. Methods: Six hundred and one BCP-ALL samples from Nordic pediatric patients (age 1-18) were CIMP classified at initial diagnosis and analyzed in relation to clinical data. Results: Among the 137 patients that later relapsed, patients with a CIMP-profile (n = 42) at initial diagnosis had an inferior overall survival (pOS(5years) 33%) compared to CIMP+ patients (n = 95, pOS(5years) 65%) (p = 0.001), which remained significant in a Cox proportional hazards model including previously defined risk factors. Conclusion: CIMP classification is a strong candidate for improved risk stratification of relapsed BCP-ALL.Peer reviewe

Efficacy of a synthetic antimicrobial peptidomimetic versus vancomycin in a Staphylococcus epidermidis device-related murine peritonitis model

Objectives: Biofilm-forming Staphylococcus epidermidis is a prevalent cause of peritonitis during peritoneal dialysis. We compared the efficacy of a synthetic antimicrobial peptidomimetic (Ltx21) versus vancomycin in a murine model mimicking a device-related peritonitis. Methods: Silicone implants, pre-colonized with an S. epidermidis biofilm, were inserted into the peritoneal cavity of BALB/c mice. Three groups (36 mice in each) with pre-colonized implants received intraperitoneal treatment with Ltx21, vancomycin or placebo. Mice were euthanized on day 3 (n¼ 12), day 6 (n¼ 12) or day 8 (n¼ 12) post-implantation. Controls were mice with sterile implants (n¼ 18) and mice without surgery (n ¼6). Bacterial reductions in cfu were analysed from implants and peritoneal fluid (PF). Inflammatory responses in serum and PF were measured. Results: Vancomycin resulted in a stronger reduction in cfu counts, both on pre-colonized implants and in PF, compared with Ltx21 and placebo. Complete bacterial clearance of the implants was not achieved in any of the groups. The implants pre-colonized with S. epidermidis 1457 resulted in a low-grade peritonitis. We observed, only on day 6, a significant increase in the PF leucocyte count in the group with pre-colonized implants compared with the group with sterile implants (P ¼ 0.0364). Conclusions: Treatment with vancomycin or Ltx21 was not sufficient to achieve complete bacterial clearance of implants, underlining the difficulties of treating such infections. The low-grade infection may attenuate the inflammatory response and contribute to impaired bacterial clearance. Keywords: biofilms, device-related peritonitis, mouse model Introduction Gram-positive bacteria, in particular coagulase-negative staphylococci, are a prevalent cause of peritoneal dialysis catheter-related peritonitis. 1 This is a serious complication that may lead to catheter removal, peritoneal membrane dysfunction and transfer to haemodialysis. 2 Peritonitis is believed to occur by bacterial entrance to the peritoneal cavity through catheter colonization. 2 Staphylococcal biofilm formation on catheters results in a reduced ability to combat such infections, both by the host immune system and by conventional treatment with antibiotics. Synthetic antimicrobial peptidomimetics (SAMPs) are novel antimicrobial agents derived from cationic antimicrobial peptides that are widespread in nature. 5 Their modes of action are not completely resolved. However, a central mechanism is bacterial membrane disruption, affecting both dormant and dividing bacteria. 7 This study aimed to investigate the efficacy of a SAMP (Ltx21) versus vancomycin in a murine model mimicking devicerelated Staphylococcus epidermidis biofilm-associated peritonitis. We assessed bacterial clearance and the host innate immune response to understand the pathophysiological mechanisms involved. Methods Bacterial isolates and MICs S. epidermidis 1457, used in this experiment, was originally isolated from a central venous catheter infection. S. epidermidis 1457 forms a thick biofilm under the in vitro growth conditions used in this study. 7 All three SAMPs have the same tripeptide sequence, with two arginine moieties providing their cationic properties and a modified tryptophan providing the lipophilic bulk. The SAMPs differ by C-terminal modifications, of which Ltx21 has an additional phenylalanine attached compared with Ltx9 and Ltx5. For Ltx21 the MIC was 6 mg/L (determined by the microbroth dilution method) and the minimal biofilm inhibitory concentration was 60 mg/L (determined by the Alamar blue method), comparable to those values previously reported for Ltx5 and Ltx9. 7 Species confirmation of small colony variants (SCVs) was performed with a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Animals and animal ethics One hundred and thirty-two female BALB/c mice (Taconic M&amp;B A/S Ry, Denmark), aged 7 -8 weeks, were used. Information on the experimental treatment of the animals and the duration of experiments (days) is provided in Device-related S. epidermidis biofilm-associated peritonitis model Silicone implants (5 mm×5 mm×2 mm; Ole Dich, Denmark) were inserted into the murine intraperitoneal cavity in order to mimic a device-related peritonitis. Briefly, the implants were prepared by incubation for 120 h in an S. epidermidis 1457 culture. The inoculum was adjusted to an optical density at 600 nm that was equivalent to that of a 2 McFarland standard in 0.9% NaCl and further suspended in tryptic soy broth (TSB) with 1% glucose to induce biofilm formation. Every 24 h the implants were rinsed in PBS and transferred to a sterile flask containing fresh medium (TSB with 1% glucose). Mice were anaethetized by subcutaneous injections (0.15 mL) in the groin area with a mixture of 0.0375 mL of 0.315 mg/mL fentanyl/10 mg/mL fluanisone (VetaPharma Ltd, UK) and 0.0375 mL of 5 mg/L midazolam (Hameln Pharmaceuticals, Germany) in 0.075 mL of sterile water. Insertion of implants and intraperitoneal treatment were performed as previously described. 9,10 Vancomycin (Sandoz, Australia) and Ltx21 (Lytix Biopharma AS, Tromsø, Norway) were both dissolved in 0.9% NaCl to a final concentration of 1 mg/mL and 0.5 mg/mL, respectively. All animals received intraperitoneal injections (400 mL) every 24 h for up to 7 days. Treatment was initiated 2 h post-implantation. The vancomycin dose was 20 mg/kg, based on previous studies. 11,12 The Ltx21 dose was 10 mg/kg, based on previous toxicology studies, a pilot treatment study and in vitro MIC studies. On the days of implant removal, mice were anaethetized by subcutaneous injection of 0.1 mL of pentobarbital (200 mg/mL) (KVL, Denmark). After general anaesthesia, blood was drawn by cardiac puncture and transferred to tubes containing heparin for fluorescence-activated cell sorting (FACS) (n¼6 animals) or 50 mg/L lepirudin (Refludan, Hoechst, Germany) for complement analysis (n¼6 animals). Peritoneal lavage was performed by injecting 5 mL of PBS into the peritoneal cavity, followed by gently massaging the abdomen before withdrawing the peritoneal fluid (PF). Implants were removed from the peritoneal cavity and transferred to tubes containing 1 mL of NaCl and 20 glass beads (Lenz, Laborglasinstrumente, Germany). Mice were euthanized by removal of the heart under general anaesthesia. Bacteriology Implants removed from the animals were vortexed for 30 s followed by 5 min of sonication at 40 kHz in an ultrasound bath (Bransonic 3510, Branso Ultrasonic Corporation, USA). One hundred microlitres of both the implant-derived suspension and PF was serial diluted and plated on blood agar plates (SSI, Denmark) for bacterial enumeration. The cfu counts were determined after incubation at 378C overnight. Prolonged incubation was necessary to detect SCVs. Cytokines, chemokines and complement Quantification of tumour necrosis factor-a (TNF-a), interleukin-1b (IL-1b), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage inflammatory protein 2 (MIP-2) and monocyte chemotactic protein-1 (MCP-1/CCL2) was performed on plasma and/or PF using the Fluorokine MAP system (R&amp;D Systems, UK) in combination with a dual-laser, flow-based sorting and detection analyser (Luminex Corporation, USA), according to the manufacturer&apos;s description. Complement factors C3a and C5a from plasma and PF were quantified using ELISA kits (USCN Life Science Inc., Wuhan, China) according to the manufacturer&apos;s instructions. Haematological parameters and flow cytometry The total leucocyte concentration and the fractions of granulocytes and macrophages were estimated in PF and blood as previously described. 13 Briefly, the fixed samples were analysed using FACS Canto (Becton Dickinson, USA). Light scatter and logarithmically amplified fluorescence parameters from at least 10 000 events were recorded in list mode after gating on forward light scatter to avoid debris, cell aggregates and bacteria. Statistics The data were analysed using GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA) or IMB SPSS Statistics 19. We used the two-way analysis of variance (ANOVA) test with Bonferroni corrections for multiple comparisons. The non-parametric Mann-Whitney U-test was Results Clinical observation Independent of treatment groups the mice exhibited signs of illness such as ruffled fur and reduced activity levels during the first 2 days. The mortality rate for mice with infected implants was 6/108 (vancomycin¼2, Ltx21¼2 and placebo¼2). The death of these mice was not anticipated, and compared with surviving mice they did not exhibit signs of illness or distress prior to death. Bacteriology Untreated pre-colonized implants had cfu counts of 3×10 8 cfu/ implant. Treatment in vivo with vancomycin and Ltx21 resulted in moderate reductions in the bacterial counts on the implants Cellular response We found no indication of an elevated systemic cellular response, either in mice with pre-colonized implants or in mice with sterile implants (data not shown). A local cellular response was observed in PF. Higher levels of leucocytes in PF from mice with pre-colonized implants receiving Ltx21, vancomycin or placebo were observed on day 6 (P ¼ 0.0364) when compared with animals with sterile implants Cytokine response Levels of TNF-a, IL-1b, GM-CSF, MIP-2 and MCP-1/CCL2 were measured in plasma and in PF on days 3, 6 and 8 post-implantation (data not shown). No significant differences were observed between the groups on any days regarding levels of IL-1b, MIP-2 and TNF-a. Mice with sterile implants showed significantly higher levels of MCP-1 in PF (P ¼ 0.005) on day 6 compared with mice with pre-colonized implants treated with placebo. GM-CSF was measured in blood only. Significantly increased values of GM-CSF were detected on days 3, 6 and 8 in mice with pre-colonized implants (P ¼0.0028) receiving Ltx21, vancomycin or placebo compared with mice with sterile implants. Significantly increased values of GM-CSF were also found on day 3 (P ¼0.02) and day 6 (P ¼ 0.04) in animals with sterile implants compared with animals without surgery. Complement activation The activation products of the complement system, C3a and C5a, were measured in plasma and PF. No significant activation of complement was observed within the groups with pre-colonized implants compared with the control groups without surgery. Discussion This murine model mimicking a device-related S. epidermidis biofilm-associated peritonitis enabled us to study the effects of two different treatment regimens and the host innate immune response. Our aim was to investigate whether Ltx21 could eradicate pre-formed S. epidermidis biofilms on peritoneal implants. Vancomycin 7 SAMPs have been found to have high levels of serum albumin binding. 14 Although in vitro time -kill kinetic studies have demonstrated rapid killing of bacteria, 6 protein binding might occur instantaneously upon administration of such peptides. 14 This reduces the amount of available peptide and might explain the reduced efficacy of Ltx21 in vivo compared with the good efficacy observed in vitro. 7,14 Vancomycin resulted in better bacterial clearance than Ltx21. However, complete biofilm clearance was not achieved by any of the two study drugs, despite high intraperitoneal dosing. On days 6 and 8 post-implantation we observed SCVs associated with implants in both treatment groups and in the placebo group. SCVs have previously been associated with persistent, subclinical and resistant infections associated with implanted medical devices. The overall low levels of granulocytes and macrophages in both blood and PF on days 3, 6 and 8 indicated a low-grade infection. Induction of macrophage apoptosis and mechanisms interfering with phagocytosis and macrophage activation has been observed for both S. epidermidis and Staphylococcus haemolyticus. 18,19 Schommer et al. 19 demonstrated that biofilm production by S. epidermidis 1457 resulted in reduced phagocytosis and macrophage activation, yielding low activation of the transcription factor NF-kB, leading to a significantly reduced IL-1b synthesis in mouse macrophage-like cells. Furthermore, in a mouse model, an S. aureus biofilm induced macrophage death and a significant reduction in IL-1b, TNF-a and MCP-1 production. 20 These observations are in line with findings from our study. In general, we found no consistent increase in cytokine production in the infected groups compared with the sterile groups. In S. epidermidis biofilm infections, a recent study reported that granulocytes are recruited and activated, but are not capable of engulfing bacteria embedded in the biofilm. No complement activation was observed in this S. epidermidis biofilm peritonitis model. In contrast, a previous study by our group demonstrated that S. epidermidis 1457 biofilm induced a strong complement activation in an ex vivo full blood model. There are limitations with this study. The model used is a suitable peritonitis model allowing simultaneous sampling of several parameters in response to treatment of an implant-associated biofilm infection. However, the current study could have benefitted from inclusion of additional animals, allowing for prolonged observation of persistence. One could argue that the use of pre-colonized implants is clinically irrelevant. However, in order to establish a biofilm infection to study the efficacy of the two different treatment regimens, we found that pre-colonization was necessary in order to obtain an infection in immunocompetent mice. Conclusions Our observations demonstrate failure of the novel SAMP Ltx21 and vancomycin in efficiently eradicating the S. epidermidis implantassociated biofilm infection. The reduced efficacy of the SAMP in vivo compared with previous in vitro results reflects the importance of performing animal studies. The presence of a persistent implant infection, which is not cleared by the innate immune system, is demonstrated. We demonstrated that this model allows for study of the complex interplay between the host immune system and the effects of antimicrobial treatment

DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia.

To access publisher's full text version of this article, please click on the hyperlink in Additional Links field or click on the hyperlink at the top of the page marked Files. This article is open access.We present a method that utilizes DNA methylation profiling for prediction of the cytogenetic subtypes of acute lymphoblastic leukemia (ALL) cells from pediatric ALL patients. The primary aim of our study was to improve risk stratification of ALL patients into treatment groups using DNA methylation as a complement to current diagnostic methods. A secondary aim was to gain insight into the functional role of DNA methylation in ALL.We used the methylation status of ~450,000 CpG sites in 546 well-characterized patients with T-ALL or seven recurrent B-cell precursor ALL subtypes to design and validate sensitive and accurate DNA methylation classifiers. After repeated cross-validation, a final classifier was derived that consisted of only 246 CpG sites. The mean sensitivity and specificity of the classifier across the known subtypes was 0.90 and 0.99, respectively. We then used DNA methylation classification to screen for subtype membership of 210 patients with undefined karyotype (normal or no result) or non-recurrent cytogenetic aberrations ('other' subtype). Nearly half (n = 106) of the patients lacking cytogenetic subgrouping displayed highly similar methylation profiles as the patients in the known recurrent groups. We verified the subtype of 20% of the newly classified patients by examination of diagnostic karyotypes, array-based copy number analysis, and detection of fusion genes by quantitative polymerase chain reaction (PCR) and RNA-sequencing (RNA-seq). Using RNA-seq data from ALL patients where cytogenetic subtype and DNA methylation classification did not agree, we discovered several novel fusion genes involving ETV6, RUNX1, and PAX5.Our findings indicate that DNA methylation profiling contributes to the clarification of the heterogeneity in cytogenetically undefined ALL patient groups and could be implemented as a complementary method for diagnosis of ALL. The results of our study provide clues to the origin and development of leukemic transformation. The methylation status of the CpG sites constituting the classifiers also highlight relevant biological characteristics in otherwise unclassified ALL patients.Swedish Foundation for Strategic Research RBc08-008 Swedish Cancer Society CAN2010/592 Swedish Childhood Cancer Foundation 11098 Swedish Research Council for Science and Technology 90559401 Swedish Research Council FORTE Swedish Research Council FORMAS Swedish Research Council VINNOVA Swedish Research Council VR 259-2012-2

Effect of Once-Weekly Azithromycin vs Placebo in Children With HIV-Associated Chronic Lung Disease: The BREATHE Randomized Clinical Trial

Importance - HIV-associated chronic lung disease (HCLD) in children is associated with small airways disease, is common despite antiretroviral therapy (ART), and is associated with substantial morbidity. Azithromycin has antibiotic and immunomodulatory activity and may be effective in treating HCLD through reducing respiratory tract infections and inflammation. Objective - To determine whether prophylactic azithromycin is effective in preventing worsening of lung function and in reducing acute respiratory exacerbations (AREs) in children with HCLD taking ART. Design, Setting, and Participants - This double-blind, placebo-controlled, randomized clinical trial (BREATHE) was conducted between 2016 and 2019, including 12 months of follow-up, at outpatient HIV clinics in 2 public sector hospitals in Malawi and Zimbabwe. Participants were randomized 1:1 to intervention or placebo, and participants and study personnel were blinded to treatment allocation. Participants included children aged 6 to 19 years with perinatally acquired HIV and HCLD (defined as forced expiratory volume in 1 second [FEV1] z score Intervention - Once-weekly oral azithromycin with weight-based dosing, for 48 weeks. Main Outcomes and Measures - All outcomes were prespecified. The primary outcome was the mean difference in FEV1 z score using intention-to-treat analysis for participants seen at end line. Secondary outcomes included AREs, all-cause hospitalizations, mortality, and weight-for-age z score. Results - A total of 347 individuals (median [interquartile range] age, 15.3 [12.7-17.7] years; 177 boys [51.0%]) were randomized, 174 to the azithromycin group and 173 to the placebo group; 162 participants in the azithromycin group and 146 placebo group participants had a primary outcome available and were analyzed. The mean difference in FEV1 z score was 0.06 (95% CI, −0.10 to 0.21; P = .48) higher in the azithromycin group than in the placebo group, a nonsignificant difference. The rate of AREs was 12.1 events per 100 person-years in the azithromycin group and 24.7 events per 100 person-years in the placebo groups (hazard ratio, 0.50; 95% CI, 0.27 to 0.93; P = .03). The hospitalization rate was 1.3 events per 100 person-years in the azithromycin group and 7.1 events per 100 person-years in the placebo groups, but the difference was not significant (hazard ratio, 0.24; 95% CI, 0.06 to 1.07; P = .06). Three deaths occurred, all in the placebo group. The mean weight-for-age z score was 0.03 (95% CI, −0.08 to 0.14; P = .56) higher in the azithromycin group than in the placebo group, although the difference was not significant. There were no drug-related severe adverse events. Conclusions and Relevance - In this randomized clinical trial specifically addressing childhood HCLD, once-weekly azithromycin did not improve lung function or growth but was associated with reduced AREs; the number of hospitalizations was also lower in the azithromycin group but the difference was not significant. Future research should identify patient groups who would benefit most from this intervention and optimum treatment length, to maximize benefits while reducing the risk of antimicrobial resistance

DNA methylation-based subtype prediction for pediatric acute lymphoblastic leukemia

Peer reviewe

Soluble biomarkers associated with chronic lung disease in older children and adolescents with perinatal HIV infection.

OBJECTIVE: HIV-associated chronic lung disease (HCLD) is a common comorbidity in children and adolescents in sub-Saharan Africa (SSA). The pathogenesis of HCLD is unclear and may be driven by underlying dysregulated systemic immune activation and inflammation. We investigated the association between 26 plasma soluble biomarkers and HCLD. DESIGN: Case--control analysis of baseline biomarker data from 336 children and adolescents (6-19 years old) with perinatal HIV infection (PHIV) and HCLD (cases) and 74 age-matched and sex-matched controls with PHIV but no CLD. HCLD was defined as having a forced expiratory volume in one second (FEV1) z score less than -1 with no reversibility. METHODS: Cryopreserved plasma collected at recruitment was used in a multiplex bead assay (Luminex) to measure baseline levels of soluble biomarkers. Logistic regression alongside data-reduction and techniques quantifying the interconnectedness of biomarkers were used to identify biomarkers associated with odds of HCLD. RESULTS: Biomarkers of general immune activation and inflammation (β2M, CRP, sCCL5, GCSF, IFN-γ, IP-10), T-cell activation (sCD25, sCD27), platelet activation (sCD40-L), monocyte activation (sCD14), coagulation (D-Dimer), cellular adhesion (E-selectin), and extracellular matrix degradation (MMP-1, MMP-7, MMP-10) were associated with increased odds of HCLD. Exploratory PCA and assessment of biomarker interconnectedness identified T-cell and platelet activation as centrally important to this association. CONCLUSION: HCLD was associated with a large number of soluble biomarkers representing a range of different pathways. Our findings suggest a prominent role for T-cell and platelet activation in HCLD

A Nationwide Study of GATA2 Deficiency in Norway-the Majority of Patients Have Undergone Allo-HSCT

PurposeGATA2 deficiency is a rare primary immunodeficiency that has become increasingly recognized due to improved molecular diagnostics and clinical awareness. The only cure for GATA2 deficiency is allogeneic hematopoietic stem cell transplantation (allo-HSCT). The inconsistency of genotype-phenotype correlations makes the decision regarding "who and when" to transplant challenging. Despite considerable morbidity and mortality, the reported proportion of patients with GATA2 deficiency that has undergone allo-HSCT is low (~ 35%). The purpose of this study was to explore if detailed clinical, genetic, and bone marrow characteristics could predict end-point outcome, i.e., death and allo-HSCT.MethodsAll medical genetics departments in Norway were contacted to identify GATA2 deficient individuals. Clinical information, genetic variants, treatment, and outcome were subsequently retrieved from the patients' medical records.ResultsBetween 2013 and 2020, we identified 10 index cases or probands, four additional symptomatic patients, and no asymptomatic patients with germline GATA2 variants. These patients had a diverse clinical phenotype dominated by cytopenia (13/14), myeloid neoplasia (10/14), warts (8/14), and hearing loss (7/14). No valid genotype-phenotype correlations were found in our data set, and the phenotypes varied also within families. We found that 11/14 patients (79%), with known GATA2 deficiency, had already undergone allo-HSCT. In addition, one patient is awaiting allo-HSCT. The indications to perform allo-HSCT were myeloid neoplasia, disseminated viral infection, severe obliterating bronchiolitis, and/or HPV-associated in situ carcinoma. Two patients died, 8 months and 7 years after allo-HSCT, respectively.ConclusionOur main conclusion is that the majority of patients with symptomatic GATA2 deficiency will need allo-HSCT, and a close surveillance of these patients is important to find the "optimal window" for allo-HSCT. We advocate a more offensive approach to allo-HSCT than previously described

Guidelines for the Diagnosis and Treatment of Meningococcal Meningitis

Meningococcal disease may present as meningitis, septicaemia or a combination of the two. Generally, meningitis has a gradual onset, with fever, headache and neck stiffness as the most frequent clinical symptoms. In contrast, fulminant septicaemia may develop within hours, and is characterised by hypotension, disseminated intravasal coagulation (DIC), petecchial bleedings and shock. Mortality with septicaemia often reaches 30%. It is of vital importance to diagnose and treat meningococcal disease rapidly. Conventionally, diagnosis is based on culture of the bacterium Neisseria meningitidis from blood or cerebrospinal fluid (CSF) or on microscopy of Gram-negative diplococci in the CSF. Direct bacterial antigen (capsular polysaccharide) detection methods have readily become available. These tests are rapid and do not require the presence of viable bacteria, but their sensitivity and specificity is low. During the last few years, a number of polymerase chain reaction (PCR) tests for the detection of bacterial nucleic acids have been developed. PCR tests are rapid, specific, extremely sensitive, does not require viable bacteria and may allow direct typing of the bacterium. The drug of choice for treating meningococcal disease is benzylpenicillin. In some rare cases, the sensitivity of N. meningitidis to penicillin is decreased, and ceftriaxone or cefotaxime may be used instead. The severe clinical signs in septicaemia are mainly caused by bacterial endotoxins which are part of the bacterial cell wall and are also released from viable bacteria. Antibiotics do not prevent the effects of endotoxins and supportive therapy to control increased intracranial pressure, hypovolaemia, DIC and shock, are also needed. Following the first case of meningococcal disease in a population, the infection may spread causing one or more secondary cases. The ideal prevention of meningococcal disease is by vaccination. However, no vaccine against group B meningococcal disease exists, and group A and C vaccines have not been implemented in most vaccination programmes. Prevention of the primary case is therefore not achievable, but secondary infection can be prevented by eradication of the disease-causing strain in healthy contacts with chemoprophylaxis.Reviews-on-treatment, Antibacterials, Bacterial-meningitis, Bacterial-meningitis, Meningococcal-vaccine-polysaccharide, Bacterial-meningitis, Reviews-on-disease, Practice-guideline, Antibacterial-vaccines