Olaf Neth

Hepatoblastoma cells express truncated neurokinin-1 receptor and can be growth inhibited by aprepitant in vitro and in vivo

BACKGROUND & AIMS Multidrug resistance presents a major problem in hepatoblastoma (HB), and new anti-tumor strategies are desperately needed. The substance P (SP)/neurokinin-1 receptor (NK1R) complex has been discovered to be pivotal in the development of a variety of human cancers, and NK1R antagonists, such as the clinical drug aprepitant, are promising future anticancer agents. Yet, the role of the SP/NK1R complex as a potential anticancer target in HB is unknown. METHODS Human HB cell lines HepT1, HepG2, and HuH6, human tumor samples from 17 children with HB as well as mice xenografted with human HB cell line HuH6 were analyzed regarding the SP/NK1R complex as a potential new anti-tumor target in HB. RESULTS Therapeutic targeting with the NK1R antagonists aprepitant, L-733,060, and L-732,138 led to growth inhibition and apoptosis in HepT1, HepG2, and HuH6 cells in a dose-dependent manner. Intriguingly, HB cells predominantly expressed the truncated splice variant of NK1R. Human fibroblasts showed only dismal NK1R expression and were significantly more resistant. Stimulation of HB cells with SP, NK1Rs natural ligand, caused increased growth rates and abrogated the anti-proliferative effect of NK1R antagonists. Expression analysis of 17 human HB samples confirmed the clinical relevance of NK1R. Most importantly, oral treatment of a HuH6 xenograft mouse model with 80mg/kg/day aprepitant for 24days resulted in a striking reduction of tumor growth, as evidenced by reduced tumor volume and weight, lowered tumor-specific alpha-fetoprotein (AFP) serum levels, and decreased number of Ki-67 positive cells. Furthermore, aprepitant treatment inhibited in vivo angiogenesis. CONCLUSIONS For the first time, we describe the NK1R in its truncated splice variant as a potent target in human HB and an inhibitory effect in vivo and in vitro by NK1R antagonists. Therefore, NK1R antagonists should be considered promising new candidates for innovative therapeutic strategies against HB.

Infant With Probable Catastrophic Antiphospholipid Syndrome Successfully Managed With Rituximab

The antiphospholipid syndrome (APS) is an acquired thrombophilic disorder characterized by the presence of autoantibodies to a variety of phospholipids and phospholipid-binding proteins. Clinical manifestations range from being asymptomatic to having imminently life-threatening events. Catastrophic antiphospholipid syndrome (CAPS) occurs in <1% of patients with APS and is defined by multiple small-vessel occlusions that lead to multiple-organ failure and is associated with high morbidity and mortality rates. Here we report the case of a 3-month-old boy with probable CAPS who presented to us with digital necrosis and pulmonary hemorrhage. In addition, a skin biopsy demonstrated multiple small-vessel thromboses without signs of vasculitis. Results of testing for autoantibodies were positive for anti-β2 glycoprotein I (anti-β2-GPI) only. His treatment consisted of high-dose steroids, immunoglobulin therapy, exchange transfusion, cyclophosphamide, and rituximab as well as iloprost and bosentan as vasodilators for his ischemia; he showed an excellent clinical response. To the best of our knowledge, this is the youngest patient with probable CAPS, the first reported patient to test positive for anti-β2-GPI antibodies and negative for anticardiolipin antibodies and lupus anticoagulant, and the second patient reported to be successfully treated with an immunomodulatory regimen including rituximab.

Reduction in external ventricular drain infection rate. Impact of a minimal handling protocol and antibiotic-impregnated catheters.

IntroductionMany strategies have been developed with the aim of reducing external ventricular drain-related infections. Antibiotic-impregnated catheters are one of them.Material and methodsWe report 648 cases of external ventricular drain from a total of 534 patients treated at the Virgen del Rocío Hospital between 1995 and 2006. Three subgroups were considered: group 1 included patients treated between 1995 and 2000, as well as a total of 190 external ventricular drains and 59 cases of infection (31.05%); group 2, with patients treated between 2000 and 2004 and managed with a minimal handling protocol, included 210 external ventricular drains and nine cases of infection (4.29%); and group 3, treated between 2004 and 2006, with 248 external ventricular drains and six cases of infection (2.41%). This latter subgroup included patients managed with a minimal handling protocol and antibiotic-impregnated catheters.ResultsInfection rate was 17% when non-antibiotic-impregnated catheters were employed and 2.41% when antibiotic-impregnated catheters were inserted (p < 0.001). This difference was statistically significant before and after the introduction of a minimal handling protocol, with percentages of 5.31% and 3.27%, respectively (p < 0.001; odds ratio 0.08; absolute risk reduction 27.26%). However, no statistically significant difference was observed in infection rate when the impact of a minimal handling protocol was considered: 4.29% when only the protocol was introduced and 2.41% when both the protocol and antibiotic-impregnated catheters were used (p > 0.05).ConclusionMinimal handling protocols constitute an essential strategy in the reduction of external ventricular drain-related infections. Besides that, the use of antibiotic-impregnated catheters may reduce infection-related hospital costs.

Neutrophils express distinct RNA receptors in a non-canonical way

Background: RNAs modulate immune responses. Neutrophils represent the major fraction of immune cells, but receptors by which neutrophils sense RNA are poorly characterized. Results: Neutrophils and differentiated HL-60 cells express the RNA receptors RIG-I, MDA-5, and TLR8. RIG-I and MDA-5 are localized in secretory vesicles. Conclusion: Neutrophils express a distinct pattern of RNA receptors. Significance: RNA receptors on neutrophils could have implications for RNA-based therapeutics. RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics.

Identifying functional defects in patients with immune dysregulation due to LRBA and CTLA-4 mutations

Heterozygous CTLA-4 deficiency has been reported as a monogenic cause of common variable immune deficiency with features of immune dysregulation. Direct mutation in CTLA-4 leads to defective regulatory T-cell (Treg) function associated with impaired ability to control levels of the CTLA-4 ligands, CD80 and CD86. However, additional mutations affecting the CTLA-4 pathway, such as those recently reported for LRBA, indirectly affect CTLA-4 expression, resulting in clinically similar disorders. Robust phenotyping approaches sensitive to defects in the CTLA-4 pathway are therefore required to inform understanding of such immune dysregulation syndromes. Here, we describe assays capable of distinguishing a variety of defects in the CTLA-4 pathway. Assessing total CTLA-4 expression levels was found to be optimal when restricting analysis to the CD45RA-Foxp3+ fraction. CTLA-4 induction following stimulation, and the use of lysosomal-blocking compounds, distinguished CTLA-4 from LRBA mutations. Short-term T-cell stimulation improved the capacity for discriminating the Foxp3+ Treg compartment, clearly revealing Treg expansions in these disorders. Finally, we developed a functionally orientated assay to measure ligand uptake by CTLA-4, which is sensitive to ligand-binding or -trafficking mutations, that would otherwise be difficult to detect and that is appropriate for testing novel mutations in CTLA-4 pathway genes. These approaches are likely to be of value in interpreting the functional significance of mutations in the CTLA-4 pathway identified by gene-sequencing approaches.

Prospective neonatal screening for severe T‐ and B‐lymphocyte deficiencies in Seville

Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X‐linked agammaglobulinemia (XLA) improves outcome of affected children. T‐cell‐receptor‐excision circles (TRECs) and kappa‐deleting‐recombination‐excision circles (KRECs) determination from dried blood spots (DBS) identify neonates with severe T‐ and/or B‐lymphopenia. No prospective data exist of the impact of gestational age (GA) and birth weight (BW) on TRECs and KRECs values.

Primer estudio piloto en España sobre el cribado neonatal de las inmunodeficiencias primarias: TRECS y KRECS identifican linfopenias T y B graves

INTRODUCTION Early diagnosis of primary immunodeficiency such as severe combined immunodeficiency (SCID) and X-linked agammaglobulinemia (XLA) improves outcome of affected infants/children. The measurement of T-cell receptor excision circles (TRECS) and kappa-deleting recombination excision circles (KRECS) can identify neonates with severe T or B-cell lymphopenia. OBJECTIVES To determine TRECS and KRECS levels from prospectively collected dried blood spot samples (DBS) and to correctly identify severe T and B-cell lymphopenia. MATERIAL AND METHODS Determination of TRECS and KRECS by multiplex PCR from neonates born in two tertiary hospitals in Seville between February 2014 and May 2014. PCR cut-off levels: TRECS<15 copies/μl, KRECS<10 copies/μl, ACTB (β-actin)>1000 copies/μl. Internal (XLA, ataxia telangiectasia) and external (SCID) controls were included. RESULTS A total of 1068 out of 1088 neonates (mean GA 39 weeks (38-40) and BW 3238g (2930-3520) were enrolled in the study. Mean (median, min/max) copies/μl, were as follows: TRECS 145 (132, 8/503), KRECS 82 (71, 7/381), and ACTB 2838 (2763, 284/7710). Twenty samples (1.87%) were insufficient. Resampling was needed in one neonate (0.09%), subsequently giving a normal result. When using lower cut-offs (TRECS<8 and KRECS<4 copies/μl), all the samples tested were normal and the internal and external controls were correctly identified. CONCLUSION This is the first prospective pilot study in Spain using TRECS/KRECS/ACTB-assay, describing the experience and applicability of this method to identify severe lymphopenias. The ideal cut-off remains to be established in our population. Quality of sampling, storage and preparation need to be further improved.

Guía de la Sociedad Española de Infectología Pediátrica para el diagnóstico y tratamiento de la toxoplasmosis congénita

Congenital toxoplasmosis is the result of transplacental fetal infection by Toxoplasma gondii after the primary maternal infection. The severity of the disease depends on the gestational age at transmission. First trimester infections are more severe, but less frequent, than third trimester infections. Acute maternal infection is diagnosed by seroconversion or by the detection of IgM antibodies and a low IgG avidity test. In these cases, spiramycin should be initiated to prevent transmission to the fetus. For identification of fetal infection, polymerase chain reaction (PCR) testing of amniotic fluid after 18 weeks gestation should be performed. If fetal infection is confirmed, the mothers should be treated with pyrimethamine, sulfadiazine and folinic acid. Most infants infected in utero are born with no obvious signs of toxoplasmosis, but up to 80% developed learning and visual disabilities later in life. Neonatal diagnosis with IgM/IgA antibodies or blood/cerebrospinal fluid PCR may be difficult because false-negative results frequently occur. In these cases diagnosis is possible by demonstrating a rise in IgG titers during follow-up or by the detection of antibodies beyond one year of age. Early treatment with pyrimethamine and sulfadiazine may improve the ophthalmologic and neurological outcome. Congenital toxoplasmosis is a preventable disease. Pre-pregnancy screening and appropriate counseling regarding prevention measures in seronegative women may prevent fetal infection.

Sequential decisions on FAS sequencing guided by biomarkers in patients with lymphoproliferation and autoimmune cytopenia

Clinical and genetic heterogeneity renders confirmation or exclusion of autoimmune lymphoproliferative syndrome difficult. To re-evaluate and improve the currently suggested diagnostic approach to patients with suspected FAS mutation, the most frequent cause of autoimmune lymphoproliferative syndrome, we prospectively determined 11 biomarkers in 163 patients with splenomegaly or lymphadenopathy and presumed or proven autoimmune cytopenia(s). Among 98 patients sequenced for FAS mutations in CD3+TCRα/β+CD4−CD8− “double negative” T cells, 32 had germline and six had somatic FAS mutations. The best a priori predictor of FAS mutations was the combination of vitamin B12 and soluble FAS ligand (cut-offs 1255 pg/mL and 559 pg/mL, respectively), which had a positive predictive value of 92% and a negative predictive value of 97%. We used these data to develop a web-based probability calculator for FAS mutations using the three most discriminatory biomarkers (vitamin B12, soluble FAS ligand, interleukin-10) of the 11 tested. Since more than 60% of patients with lymphoproliferation and autoimmune cytopenia(s) in our cohort did not harbor FAS mutations, 15% had somatic FAS mutations, and the predictive value of double-negative T-cell values was rather low (positive and negative predictive values of 61% and 77%, respectively), we argue that the previously suggested diagnostic algorithm based on determination of double-negative T cells and germline FAS sequencing, followed by biomarker analysis, is not efficient. We propose vitamin B12 and soluble FAS ligand assessment as the initial diagnostic step with subsequent decision on FAS sequencing supported by a probability-calculating tool.

Successful management of chronic multifocal Q fever Osteomyelitis with adjuvant interferon-gamma therapy.

We present a 3-year-old girl who had chronic recurrent multifocal osteomyelitis caused by Coxiella burnetii despite long-term dual antibiotic therapy. Excellent clinical response was achieved and sustained when immunomodulatory therapy with interferon-γ was initiated. This is the case of a first child who was successfully treated with interferon-γ as adjuvant therapy for chronic multifocal Q fever osteomyelitis.