Wojciech Mlynarski

Administration of CD4+CD25highCD127− Regulatory T Cells Preserves β-Cell Function in Type 1 Diabetes in Children

OBJECTIVE Type 1 diabetes is a condition in which pancreatic islets are destroyed by self-reactive T cells. The process is facilitated by deficits in the number and suppressive activity of regulatory T cells (Tregs). Here, we show for the first time that the infusion of autologous Tregs prolongs remission in recently diagnosed type 1 diabetes in children. RESEARCH DESIGN AND METHODS We have administered Tregs in 10 type 1 diabetic children (aged 8–16 years) within 2 months since diagnosis. In total, 4 patients received 10 × 106 Tregs/kg body wt, and the remaining 6 patients received 20 × 106 Tregs/kg body wt. The preparation consisted of sorted autologous CD3+CD4+CD25highCD127− Tregs expanded under good manufacturing practice conditions. RESULTS No toxicity of the therapy was noted. A significant increase in the percentage of Tregs in the peripheral blood has been observed since the day of infusion. These patients were followed along with matched type 1 diabetic patients not treated with Tregs. Half a year after type 1 diabetes onset (4–5 months after Tregs infusion), 8 patients treated with Tregs still required <0.5 UI/kg body wt of insulin daily, with 2 patients out of insulin completely, whereas the remission was over in the nontreated group. In addition, plasma C-peptide levels were significantly higher in the treated group as compared with those not treated. CONCLUSIONS This study shows that the administration of Tregs is safe and tolerable in children with recent-onset type 1 diabetes.

Functional C3435T polymorphism of MDR1 gene: an impact on genetic susceptibility and clinical outcome of childhood acute lymphoblastic leukemia.

The significance of genetic background in childhood acute lymphoblastic leukemia (ALL) is not well understood. Polymorphisms of genes encoding for xenobiotics and drug transporters are potential factors, which can influence the risk of developing ALL and its clinical outcome. P‐glycoprotein (P‐gp) is an adenosine triphosphate‐binding cassette (ABC)‐family transporter involved in protection against xenobiotics and multi‐drug resistance. Recently, the single‐nucleotide polymorphism C3435T of MDR1 gene has been found to be associated with altered tissue expression and function of P‐gp. To evaluate whether C3435T MDR1 polymorphism is associated with the occurrence and outcome of ALL, 113 children with ALL (median age 5.1 yr) and 175 healthy individuals of Polish Caucasian origin were studied by polymerase chain reaction‐restriction fragment‐length polymorhism (PCR‐RFLP) assay. The mutant homozygous TT genotype was found to be associated with occurrence of ALL (OR, 95% CI; 1.8, 1.1–3.1; P = 0.037). Besides, the analysis of factors influencing clinical outcome of our ALL patient cohort showed that CC genotype carriers had significantly lower event‐free survival probability (pEFS) (0.62 vs. 0.87; P = 0.007) and overall survival probability (pOS) (0.72 vs. 0.91; P = 0.006). The Cox proportional hazards model‐based analysis revealed that the hazard ratios for lower pEFS and lower pOS among CC homozygous subjects were 3.9 (P = 0.008) and 3.3 (P = 0.02), respectively. In conclusion, the results of the present study provide evidence that C3435T MDR1 polymorphism may involve both the susceptibility to and the clinical outcome of childhood ALL. Carriers of the TT genotype are more at risk of developing ALL than other individuals, whereas CC genotype carriers are supposed to have worse prognosis.

Therapy of type 1 diabetes with CD4+CD25highCD127-regulatory T cells prolongs survival of pancreatic islets — Results of one year follow-up

It is hypothesized that CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) can prevent destruction of pancreatic islets protecting from type 1 diabetes (DM1). Here we present results of one year follow-up of 12 DM1 children treated with autologous expanded ex vivo Tregs. Patients received either a single or double Tregs infusion up to the total dose of 30×10(6)/kg. No severe adverse effects were observed. The treatment did not impair post-immunization antibody responses. Tregs infusion was followed by increase in Tregs number in peripheral blood. Most of the patients responded to the therapy with increase in C-peptide levels (8/12 and 4/6 after the first and the second dose, respectively). Tregs administration resulted also in lower requirement for exogenous insulin (8/12 treated patients versus 2/10 untreated controls in remission) with two children completely insulin independent at one year. Repetitive administration of Tregs is safe and can prolong survival of β-cells in DM1 (registration: ISRCTN06128462).

Mutations at the BLK locus linked to maturity onset diabetes of the young and β-cell dysfunction

Maturity-onset diabetes of the young (MODY) is a subtype of diabetes defined by an autosomal pattern of inheritance and a young age at onset, often before age 25. MODY is genetically heterogeneous, with 8 distinct MODY genes identified to date and more believed to exist. We resequenced 732 kb of genomic sequence at 8p23 in 6 MODY families unlinked to known MODY genes that showed evidence of linkage at that location. Of the 410 sequence differences that we identified, 5 had a frequency <1% in the general population and segregated with diabetes in 3 of the families, including the 2 showing the strongest support for linkage at this location. The 5 mutations were all placed within 100 kb corresponding to the BLK gene. One resulted in an Ala71Thr substitution; the other 4 were noncoding and determined decreased in vitro promoter activity in reporter gene experiments. We found that BLK—a nonreceptor tyrosine-kinase of the src family of proto-oncogenes—is expressed in β-cells where it enhances insulin synthesis and secretion in response to glucose by up-regulating transcription factors Pdx1 and Nkx6.1. These actions are greatly attenuated by the Ala71Thr mutation. These findings point to BLK as a previously unrecognized modulator of β-cell function, the deficit of which may lead to the development of diabetes.

ISPAD Clinical Practice Consensus Guidelines 2014. The diagnosis and management of monogenic diabetes in children and adolescents.

Oscar Rubio-Cabezasa, Andrew T Hattersleyb, Pål R Njølstadc,d, Wojciech Mlynarskie, Sian Ellardb, Neil Whitef, Dung Vu Chig and Maria E Craigh,i aDepartment of Paediatric Endocrinology, Hospital Infantil Universitario Niño Jesús, Madrid, Spain; bInstitute of Biomedical and Clinical Sciences, University of Exeter Medical School, Exeter, UK; cDepartment of Clinical Science, University of Bergen, Bergen, Norway; dDepartment of Pediatrics, Haukeland University Hospital, Bergen, Norway; eDepartment of Pediatrics, Oncology, Hematology and Diabetology, Medical University of Lodz, Lodz, Poland; fDivision of Pediatric Endocrinology and Metabolism, Department of Pediatrics, Washington University School of Medicine, St Louis Children’s Hospital, St. Louis, MO, USA; gDepartment of Pediatric Endocrinology, National Hospital for Pediatrics, Hanoi, Vietnam; hThe Children’s Hospital at Westmead and Discipline of Pediatrics and Child Health, University of Sydney, Sydney, Australia and iSchool of Women’s and Children’s Health, University of New South Wales, Sydney, Australia

Sulfonylurea improves CNS function in a case of intermediate DEND syndrome caused by a mutation in KCNJ11

Background A 12-week-old female presented with neonatal diabetes. Insulin therapy alleviated the diabetes, but the patient showed marked motor and mental developmental delay. The patient underwent genetic evaluation at the age of 6 years, prompted by reports that mutations in the KCNJ11 gene caused neonatal diabetes.Investigations Genomic sequencing of the ATP-sensitive potassium (KATP) channel gene KCNJ11 and in vitro functional analysis of the channel defect, and single-photon emission CT imaging before and after glibenclamide therapy.Diagnosis Genetic evaluation revealed a missense mutation (His46Leu) in KCNJ11, which encodes the Kir6.2 subunit of the KATP channel, conferring reduced ATP sensitivity. Functional studies demonstrated that the mutant channels were strongly inhibited by the sulfonylurea tolbutamide.Management Sulfonylurea (glibenclamide) treatment led to both improved glucose homeostasis and an increase in mental and motor function.

Insulin sensitivity in Type 1 diabetic children and adolescents.

Aim  To estimate insulin sensitivity in Type 1 diabetic children and adolescents, and assess the relationship between insulin sensitivity and clinical markers of adiposity and parameters of the metabolic syndrome.

The anti-asparagines antibodies correlate with l-asparagines activity and may affect clinical outcome of childhood acute lymphoblastic leukemia

The primary aim of the study was to evaluate the importance of anti-asparaginase antibodies for l-asparaginase activity in children with standard and medium risk acute lymphoblastic leukemia (ALL). Forty-seven children with newly diagnosed ALL were included into the prospective study. Enzyme activity and the presence of anti-asparaginase antibodies (IgG and IgM class) were determined. Anti-asparaginase antibodies were identified in 13/47 (IgM class) and 10/47 (IgG class) patients in the induction and in 19/47 (IgM class) and 20/47 (IgG class) patients in the reinduction phase of treatment. The enzyme activity was lower in patients that were positive for anti-asparaginase antibodies, especially in reinduction phase (median 37 (20 – 180) vs 355 (141 – 499), p = 0.001). An association between anti-asparaginase antibodies and the allergic reaction to the drug was found. Besides, the children who developed anti-asparaginase antibodies in the induction phase of treatment showed lower event-free survival as well as overall survival in comparison with children without antibodies. Since our study was carried out in a small number of patients, this observation is only speculative and needs to be confirmed by a further study on a larger sample size, with multivariable analysis. However, our data suggest that l-asparaginase activity together with anti-asparaginase antibodies measurements may become useful for effective therapy of ALL.

Genetic polymorphisms in DNA base excision repair gene XRCC1 and the risk of squamous cell carcinoma of the head and neck

BackgroundThe genes of base excision repair (BER) pathway have been extensively studied in the association with various human cancers. We performed a case-control study to test the association between two common single nucleotide polymorphisms (SNPs) of XRCC1 gene with human head and neck squamous cell carcinoma (HNSCC).MethodsThe genotype analysis of Arg194Trp and Arg399Gln gene polymorphisms for 92 HNSCC patients and 124 controls of cancer free subjects, in Polish population were performed using the PCR-based restriction fragment length polymorphism (PCR-RFLP) with endonuclease Msp I.ResultsNo altered risk has been found individually for these SNPs, however haplotypes analysis showed high association with head and neck cancer. The highest frequency, according to wild-type of Arg194Arg and Arg399Arg genotypes, was identified for Arg194Trp-Arg399Arg haplotype (OR, 2.96; 95% CI, 1.01–8.80).ConclusionFinally, we identified the combined Arg194Trp-Arg399Arg genotype of base excision repair gene XRCC1 that was associated with HNSCC and may have an impact on identification of a high-risk cancer population.

The cross-reactivity of anti-asparaginase antibodies against different L-asparaginase preparations.

Repeated administration of l-asparaginase leads to the development of specific antibodies and hypersensitivity reactions. The aim of the study was to evaluate a possible cross-reaction of anti-asparaginase antibodies, developed against the native E. colil-asparaginase (Asparaginase Medac), with other preparations of the enzyme. Sixteen patients with acute lymphoblastic leukemia, in whom in the reinduction phase of treatment hypersensitivity against l-asparaginase was observed and/or the presence of anti-asparaginase antibodies was established were recruited for the present study. Ten out of 16 tested sera showed cross-immunoreactivity to PEG-asparaginase, while no reactivity to l-asparaginase derived from Erwinia chrysantemi was observed. Since cross-reacting antibodies were also found in sera of patients with no overt allergic reaction, l-asparaginase may undergo silent inactivation during the reinduction phase of therapy. This finding is of clinical importance with regard to appropriate dosage and necessitates careful enzyme activity monitoring in all patients undergoing repeated treatment with various l-asparaginase preparations.