Norman Händel

Autoimmunity, Intestinal Lymphoid Hyperplasia, and Defects in Mucosal B-Cell Homeostasis in Patients With PTEN Hamartoma Tumor Syndrome

The Phosphatase And Tensin Homolog Deleted On Chromosome 10 (PTEN) regulates the phosphoinositol-3-kinase (PI3K)-AKT signaling pathway. In a series of 34 patients with PTEN mutations, we described gastrointestinal lymphoid hyperplasia, extensive hyperplastic tonsils, thymus hyperplasia, autoimmune lymphocytic thyroiditis, autoimmune hemolytic anemia, and colitis. Functional analysis of the gastrointestinal mucosa-associated lymphoid tissue revealed increased signaling via the PI3K-AKT pathway, including phosphorylation of S6 and increased cell proliferation, but also reduced apoptosis of CD20(+)CD10(+) B cells. Reduced activity of PTEN therefore affects homeostasis of human germinal center B cells by increasing PI3K-AKT signaling via mammalian target of rapamycin as well as antiapoptotic signals.

Sirolimus treatment of severe PTEN hamartoma tumor syndrome: case report and in vitro studies

Background:Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is caused by germ line mutations in the PTEN gene. Symptoms include cancer predisposition, immune deviations, and lipomas/lipomatosis. No causal standard therapy is available. We describe a therapeutic attempt with the mammalian target of rapamycin (mTOR) inhibitor sirolimus for a PHTS patient suffering from thymus hyperplasia and lipomatosis. We furthermore assessed the in vitro effects of sirolimus and other inhibitors on lipoma cells of the patient.Methods:The patient underwent clinical and blood examinations and whole-body magnetic resonance imaging to assess tumor sizes. Lipoma cells of the patient were incubated with inhibitors of the phosphoinositide-3-kinase (PI3K)/AKT/mTOR signaling pathway to analyze the effects on proliferation, adipocyte differentiation, and survival in vitro.Results:Sirolimus treatment improved somatic growth and reduced thymus volume. These effects diminished over the treatment period of 19 mo. Sirolimus decreased lipoma cell proliferation and adipocyte differentiation in vitro but did not cause apoptosis. PI3K and AKT inhibitors induced apoptosis significantly.Conclusion:Sirolimus treatment led to an improvement of the patient’s clinical status and a transient reduction of the thymus. Our in vitro findings point to PI3K and AKT inhibitors as potential treatment options for patients with severe forms of PHTS.

Immune dysregulation in patients with PTEN hamartoma tumor syndrome: Analysis of FOXP3 regulatory T cells

Background: Patients with heterozygous germline mutations in phosphatase and tensin homolog deleted on chromosome 10 (PTEN) experience autoimmunity and lymphoid hyperplasia. Objectives: Because regulation of the phosphoinositide 3‐kinase (PI3K) pathway is critical for maintaining regulatory T (Treg) cell functions, we investigate Treg cells in patients with heterozygous germline PTEN mutations (PTEN hamartoma tumor syndrome [PHTS]). Methods: Patients with PHTS were assessed for immunologic conditions, lymphocyte subsets, forkhead box P3 (FOXP3)+ Treg cell levels, and phenotype. To determine the functional importance of phosphatases that control the PI3K pathway, we assessed Treg cell induction in vitro, mitochondrial depolarization, and recruitment of PTEN to the immunologic synapse. Results: Autoimmunity and peripheral lymphoid hyperplasia were found in 43% of 79 patients with PHTS. Immune dysregulation in patients with PHTS included lymphopenia, CD4+ T‐cell reduction, and changes in T‐ and B‐cell subsets. Although total CD4+FOXP3+ Treg cell numbers are reduced, frequencies are maintained in the blood and intestine. Despite pathogenic PTEN mutations, the FOXP3+ T cells are phenotypically normal. We show that the phosphatase PH domain leucine‐rich repeat protein phosphatase (PHLPP) downstream of PTEN is highly expressed in normal human Treg cells and provides complementary phosphatase activity. PHLPP is indispensable for the differentiation of induced Treg cells in vitro and Treg cell mitochondrial fitness. PTEN and PHLPP form a phosphatase network that is polarized at the immunologic synapse. Conclusion: Heterozygous loss of function of PTEN in human subjects has a significant effect on T‐ and B‐cell immunity. Assembly of the PTEN‐PHLPP phosphatase network allows coordinated phosphatase activities at the site of T‐cell receptor activation, which is important for limiting PI3K hyperactivation in Treg cells despite PTEN haploinsufficiency. GRAPHICAL ABSTRACT Figure. No caption available.

A recessive form of extreme macrocephaly and mild intellectual disability complements the spectrum of PTEN hamartoma tumour syndrome

PTEN hamartoma tumour syndrome (PHTS) is caused by heterozygous variants in PTEN and is characterised by tumour predisposition, macrocephaly, and cognition impairment. Bi-allelic loss of PTEN activity has not been reported so far and animal models suggest that bi-allelic loss of PTEN activity is embryonically lethal. Here, we report the identification of a novel homozygous variant in PTEN, NM_000314.4; c.545T>C; p.Leu182Ser, in two adolescent siblings with severe macrocephaly and mild intellectual disability. The variant is predicted to be damaging and is associated with significantly increased phospho-S6 downstream of PTEN. The absence of tumours in the two homozygous siblings as well as lack of symptoms of PHTS in the heterozygous carriers of the family suggest that this particular variant is functionally hypomorphic rather than deleterious.

Resveratrol Potentiates Growth Inhibitory Effects of Rapamycin in PTEN-deficient Lipoma Cells by Suppressing p70S6 Kinase Activity.

ABSTRACT Patients with phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome and germline mutations in PTEN frequently develop lipomatosis, for which there is no standard treatment. Rapamycin was shown to reduce the growth of lipoma cells with heterozygous PTEN deficiency in vitro, but concomitantly induced an upregulation of AKT phosphorylation. Since it was shown that resveratrol stabilizes PTEN, we asked whether co-incubation with resveratrol could suppress the rapamycin-induced AKT phosphorylation in PTEN-deficient lipoma cells. Resveratrol incubation resulted in decreased lipoma cell viability by inducing G1-phase cell cycle arrest and apoptosis. PTEN expression and AKT phosphorylation were not significantly changed, whereas p70S6 kinase (p70S6K) phosphorylation was reduced in PTEN-deficient lipoma cells after resveratrol incubation. Rapamycin/resveratrol co-incubation significantly decreased viability further at lower doses of resveratrol and resulted in decreased p70S6K phosphorylation compared to rapamycin incubation alone, suggesting that resveratrol potentiated the growth inhibitory effects of rapamycin by reducing p70S6K activation. Both viability and p70S6K phosphorylation of primary PTEN wild-type preadipocytes were less affected compared to PTEN-deficient lipoma cells by equimolar concentrations of resveratrol. These results support the concept of combining chemopreventive natural compounds with mammalian target of rapamycin (mTOR) inhibitors to increase the efficacy of chemotherapeutic drugs for patients suffering from overgrowth syndromes.

Cell-cell-neighborhood relations in tissue sections--a quantitative model for tissue cytometry.

Physical interactions between different cell types are a requirement for the initiation and maintenance of immune responses. The distribution pattern of cells within a tissue may result from specific cell‐cell‐interactions or random distribution. Tissue architecture, degree of inflammation, frequencies of cells, number of contact partners, cell type, and size as well as cell movement and contact time determine the distribution of cells within tissues. We developed a matrix model to determine the degree of expected random distribution of two cell types (A and B) and cell‐cell‐contacts within tissue sections. The model predictions were compared with experimental data derived from immunofluorescence microscopy. We implemented a computer algorithm for automatic image analysis to visualize and quantify cell‐cell‐neighborhood relations. Using the number of cells type A (a), the total cell number (t) and the mean number of cells that are in contact with cells type B (cB), the ratio of cells type B in contact with cells type A can be described by bA/b = 1− (1− (a/t))⁁ˆcB. We applied the model system to investigate the distribution of Foxp3+ regulatory T cells with Ki‐67+ proliferating cells within mouse tissue sections. The matrix model provides a tool to describe the expected distribution of two different cell types and their cell‐cell‐contacts within tissues. Comparing the degree of expected random distribution with experimental data might help to propose functional cell‐cell‐interactions in tissue sections.

New pediatric percentiles of liver enzyme serum levels (alanine aminotransferase, aspartate aminotransferase, γ‐glutamyltransferase): Effects of age, sex, body mass index, and pubertal stage

The present study aims to clarify the effects of sex, age, body mass index (BMI), and puberty on transaminase serum levels in children and adolescents and to provide new age‐ and sex‐related percentiles for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ‐glutamyltransferase (GGT). Venous blood and anthropometric data were collected from 4,126 cases. Excluded were cases of participants with potential hepatotoxic medication, with evidence of potential illness at the time of blood sampling and non‐normal BMI (BMI <10th or >90th). The resulting data (N = 3,131 cases) were used for the calculations of ALT, AST, and GGT percentiles. Age‐ and sex‐related reference intervals were established by using an LMS method of Cole–type method. Serum levels of transaminases follow age‐specific patterns and relate to the onset of puberty. This observation is more pronounced in girls than in boys. ALT percentiles showed similar‐shaped patterns in both sexes. Multivariate regression confirmed significant effects of puberty and BMI‐SDS (β = 2.21) on ALT. Surprisingly, AST serum levels were negatively influenced by age (β = −1.42) and BMI‐SDS (β = −0.15). GGT percentiles revealed significant sex‐specific differences, correlated positively with age (β = 0.37) and showed significant association with BMI‐SDS (β = 1.16). Conclusion: Current reference values of ALT, AST, and GGT serum levels were calculated for children between 11 months and 16.0 years, using modern analytical and statistical methods. This study extends the current knowledge about transaminases by revealing influences of age, sex, BMI, and puberty on serum concentrations of all three parameters and has for these parameters one of the largest sample sizes published so far. (Hepatology 2017).

Antinuclear Antibody-Positive Juvenile Idiopathic Arthritis Despite IRAK-4 Deficiency

To the editor, Juvenile idiopathic arthritis (JIA) is an antigen-driven autoimmune disease with abnormalities in the adaptive immune system [1]. Deficiency of the interleukin-1 receptor-associated kinase (IRAK)-4 (OMIM #607676) is an autosomal recessive, rare disorder of the innate immunity, with a predominant susceptibility to invasive infections with pyogenic bacteria [2]. Changes in signaling via the toll-like receptors (TLR) lead to diminished nuclear translocation of NF-κB and inhibition of transcription of various inflammatory genes [3]. While patients with IRAK-4 deficiency accumulate autoreactive B cells in the blood, the inhibition of the TLR signaling pathway is thought to make autoimmunity impossible. So far, no autoimmune disease has been reported in affected patients [4]. Here, we present the case of a patient with antinuclear antibody (ANA)-positive juvenile idiopathic arthritis with genetically confirmed, fatal IRAK-4 deficiency. The patient was a 3-year-old Caucasian girl who presented with active arthritis of the right knee and left ankle joint following two episodes of invasive streptococcal skin infections. She was found to have a positive titer of ANA (1:160) and was rheumatoid factor and HLA-B27 negative. Elevated ANA titers up to 1:320 were also found on subsequent time points, but no subtyping of specific antibodies was performed. Family history showed no other cases of autoimmune disease, but notably, a brother who had died at the age of 7 months due to septic shock following confirmed pneumococcal infection; he also had lymph node abscesses that had to be treated surgically at the age of 4 months. She was diagnosed with ANApositive juvenile idiopathic arthritis, category oligoarthritis according to ILAR criteria, and treated with ibuprofen and sulfasalazine [5]. Due to ongoing arthritis, she was switched to methotrexate after 5 months and went into complete remission for 24 months. Investigations due to recurrent gastrointestinal symptoms also demonstrated Salmonella carrier status over a period of 9 months, preceding the onset of arthritis for over 6 months. She had received all recommended vaccinations according to the national schedule including conjugate vaccinations against pneumococcus. She presented again, still on treatment with methotrexate, at the age of 5 years with high fever, headache, and recurrent vomiting. On examination, she was found to have left-sided acute otitis media and mastoiditis. She rapidly progressed to septic shock, with seizures and respiratory insufficiency. An infectious workup demonstrated only highly positive findings for pneumococcal antigen in the urine; serum immunoglobulin and complement levels were normal, and vaccination antibodies showed normal results. No further immunological workup was performed. CT showed meningoencephalitis and signs of elevated intracerebral pressure; she developed multi-organ failure and died on the third day after admission due to tonsillar herniation. Subsequent genetic analysis showed a homozygous c.C877T (p.Q293X) mutation in exon 6 of the IRAK-4 gene in both the patient and her younger brother, where genetic material was still available. IRAK-4 is a component in the TLR/interleukin-1 pathway, which activates nuclear factor κB (NF-κB) and mitogenactivated protein kinases (MAPKs) via the IRAK complex, together with IRAK-1 [3]. Activation of NF-κB and MAPK then leads to binding of downstream molecules (AP-1, p50/ p65 complex) to DNA at cognate binding sites and regulation of gene transcription. Autosomal recessive IRAK-4 * Volker Schuster schv@medizin.uni–leipzig.de

Simvastatin induces apoptosis in PTEN‑haploinsufficient lipoma cells

Adipose tissue tumors (lipomas) frequently develop in patients with heterozygous germ line phosphatase and tensin homolog (PTEN) mutations. simvastatin has been demonstrated to exhibit antitumor effects, and so the aim of the present study was to assess the effects of simvastatin on the growth of human PTEN haploinsufficient lipoma cells. Whether the effects of simvastatin in lipomas are mediated via PTEN upregulation was also assessed. The results of the present study revealed that simvastatin treatment reduced cell viability and induced apoptosis in human lipoma cells. Furthermore, it was demonstrated that the expression of cellular PTEN mRNA and protein was increased following simvastatin stimulation. In addition, the phosphorylation of protein kinase B and downstream targets of mammalian target of rapamycin and 4E‑binding protein (4E‑BP)‑1 was attenuated. It was also demonstrated that simvastatin induced PTEN transcriptional upregulation by increasing peroxisome proliferator‑activated receptor (PPAR)γ expression. The small interfering RNA‑mediated knockdown of PPARγ abrogated the stimulatory effect of simvastatin on the PTEN protein, but did not influence apoptosis. The results of the present study suggest that simvastatin may be beneficial for patients with inoperable PTEN haploinsufficient lipomas.

Antibody Concentrations Decrease 14-Fold in Children With Celiac Disease on a Gluten-Free Diet but Remain High at 3 Months

Background & Aims Celiac disease can be identified by a serologic test for IgA against tissue transglutaminase (IgA‐TTG) in a large proportion of children. However, the increased concentrations of antibody rarely normalize within the months after children are placed on a gluten‐free diet (GFD). Early serologic predictors of sufficient adherence to GFD are required for optimal treatment. Methods In a prospective study, we observed the response to a GFD in 345 pediatric patients (67% girls; mean age, 8.4 y) who underwent duodenal biopsy to confirm or refute celiac disease from October 2012 through December 2015. Baseline serum samples were tested centrally for IgA‐TTG and IgG against deamidated gliadin. Follow‐up serologic analyses of children on a GFD were performed about 3 months later. Results The geometric mean concentration of IgA‐TTG decreased from 72.4‐fold to 5.2‐fold the upper limit of normal (ULN), or by a factor of 14.0 (95% CI, 12.0–16.4). A substantial response (defined as a larger change than the typical variation in patients not on a GFD) was observed in 80.6% of the children. Only 28.1% of patients had a substantial response in the concentration of IgG against deamidated gliadin. Concentration of IgA‐TTG remained above 1‐fold the ULN in 83.8% of patients, and above 10‐fold the ULN in 26.6% of patients with a substantial response. Conclusions Serum concentration of IgA‐TTG decreases substantially in most children with celiac disease within 3 months after they are placed on a GFD, but does not normalize in most. This information on changes in antibody concentrations can be used to assess patient response to the diet at short‐term follow‐up evaluations. Patients with a substantial response to a GFD often still have high antibody levels after 3 months. German Clinical Trials Registry no. DRKS00003854.