Rapamycin-based graft-versus-host disease prophylaxis increases the immunosuppressivity of myeloid-derived suppressor cells without affecting T cells and anti-tumor cytotoxicity.

The immunosuppressant rapamycin (RAPA) inhibits mammalian target of rapamycin (mTOR) functions and is applied after allogeneic bone marrow transplantation (BMT) to attenuate the development of graft-versus-host disease (GVHD), although the cellular targets of RAPA treatment are not well defined. Allogeneic T cells are the main drivers of GVHD, while immunoregulatory myeloid-derived suppressor cells (MDSCs) were recently identified as potent disease inhibitors. In this study, we analyzed whether RAPA prevents the deleterious effects of allogeneic T cells or supports the immunosuppressive functions of MDSCs in a BMT model with major histocompatibility complex (MHC) classes I and II disparities. RAPA treatment efficiently attenuated clinical and histological GVHD and strongly decreased disease-induced mortality. Although splenocyte numbers increased during RAPA treatment, the ratio of effector T cells to MDSCs was unaltered. However, RAPA treatment induced massive changes in the genomic landscape of MDSCs preferentially up-regulating genes responsible for uptake or signal transduction of lipopeptides and lipoproteins. Most importantly, MDSCs from RAPA-treated mice exhibited increased immunosuppressive potential, which was primarily inducible nitric oxide synthase (iNOS)-dependent. Surprisingly, RAPA treatment had no impact on the genomic landscape of T cells, which was reflected by unchanged expression of activation and exhaustion markers and cytokine profiles in T cells from RAPA-treated and untreated mice. Similarly, T cell cytotoxicity and the graft-versus-tumor effect were maintained as co-transplanted tumor cells were efficiently eradicated, indicating that the immunosuppressant RAPA might be an attractive approach to strengthen the immunosuppressive function of MDSCs without affecting T cell immunity.

Prevalence and phenotypic characterization of MC4R variants in a large pediatric cohort.

OBJECTIVE: We aimed to determine the prevalence of melanocortin-4 receptor (MC4R) variants in a large German cohort of children with obesity in a pediatric outpatient clinic and to ascertain whether there is a specific phenotype associated with loss-of-function variants as previously reported. STUDY DESIGN: Eight hundred and ninety-nine patients from our pediatric obesity clinic were screened for MC4R variants by DNA sequencing after PCR amplification. Retrospective statistical analysis of anthropometric and metabolic characteristics was performed, comparing patients with and without MC4R variants across the entire cohort (n=586) as well as in case-control analysis using patients with common sequence MC4R individually matched for age, sex and body mass index standard deviation score (SDS) (n=11 case-control pairs). RESULTS: We identified heterozygous variants within the coding region of the MC4R gene in n=22 (2.45%) patients. Fourteen (1.56%) had a variant that impaired receptor function. One new frameshift (p.F152Sfs), an yet unpublished nonsense mutation (p.Q156X) and one nonsynonymous variation (p.V65E) described in the Mouse Genome Database were detected. Across the whole cohort, at all ages, mean height SDS in subjects with impaired receptor function was higher than in patients with common sequence MC4R. In matched individuals, this trend persisted (8 of the 11 pairs) within the case-control setting. No differences were found regarding metabolic characteristics. CONCLUSIONS: The observed prevalence of mutations causing impaired receptor function in this large cohort is comparable to other pediatric cohorts. MC4R deficiency tends to lead to a taller stature, confirming previous clinical reports. The association of MC4R mutations with a distinct phenotype concerning metabolic characteristics remains questionable.

Simultaneous Interference with HER1/EGFR and RAC1 Signaling Drives Cytostasis and Suppression of Survivin in Human Glioma Cells in Vitro.

We have previously reported that combined inhibition of the epidermal growth factor receptor by erlotinib and of RAC1 by NSC23766 yielded a synergistic antiproliferative effect on established and primary cultured glioblastoma cells. The current study aimed at identifying the molecular mechanism. Staining for annexin V/PI or carboxyfluorescein succinimidyl ester was performed in order to determine the induction of apoptosis, necrosis or cytostasis in established and primary cultured glioblastoma cells. Moreover, expression of Ki-67 was determined by immunofluorescence, and the expression of cell cycle proteins was analysed by Western blot. Our data show that combined treatment with erlotinib and NSC23766 resulted in a reduced number of cell divisions, a significantly decreased Ki-67 expression, increased apoptosis and autophagy when compared to single agent treatments. On the molecular level, concomitant treatment with both agents resulted in a pronounced downregulation of cyclin D1, cyclin-dependent kinases 2, 4 and 6, as well as of survivin when compared to treatments with either agent alone. In conclusion, we demonstrate that combined treatment of human glioma cell lines in vitro with erlotinib and NSC23766 markedly inhibits cell division, induces apoptosis independent of caspase-3 activation and induces autophagy concomitant with suppression of survivin.

The Cause of Acute Respiratory Failure Predicts the Outcome of Noninvasive Ventilation in Immunocompromised Children.

BACKGROUND: Noninvasive ventilation (NIV) may be superior to conventional therapy in immunocompromised children with respiratory failure. METHODS: Mortality, success rate, prognostic factors and side effects of NIV for acute respiratory failure (ARF) were investigated retrospectively in 41 in children with primary immunodeficiency, after stem cell transplantation or chemotherapy for oncologic disease. RESULTS: In 11/41 (27%) children invasive ventilation was avoided and patients were discharged from ICU. In children with NIV failure ICU-mortality was 19/30 (63%). 8/11 (72%) children with NIV success had recurrence of ARF after 27 days. Only 4/11 (36%) children with first episode NIV success and 8/30 (27%) with NIV failure survived to hospital discharge. Lower FiO2, SpO2/FiO2 and blood culture positive bacterial sepsis were predictive for NIV success, while fungal sepsis or culture negative ARF were predictive for NIV failure. We observed catecholamine treatment in 14/41 (34%), pneumothorax in 2/41 (5%), mediastinal emphysema in 3/41 (7%), a life threatening nasopharyngeal hemorrhage and need for resuscitation during intubation in 5/41 (12%) NIV-episodes. CONCLUSIONS: The prognosis of ARF in immunocompromised children remains guarded independent of initial success or failure of NIV due to a high rate of recurrent ARF. Reversible causes like bacterial sepsis had a higher NIV response rate. Relevant side effects of NIV were observed.

Key treatment questions in childhood acute lymphoblastic leukemia: results in 5 consecutive trials performed by the ALL-BFM study group from 1981 to 2000.

Between 1981 and 2000, 6 609 children (<18 years of age) were treated in 5 consecutive trials of the Berlin-Frankfurt-Münster (BFM) study group for childhood acute lymphoblastic leukemia (ALL). Patients were treated in up to 82 centers in Germany, Austria, and Switzerland. Probability of 10-year event-free survival (survival) improved from 65% (77%) in study ALL-BFM 81-78% (85%) in ALL-BFM 95. In parallel to relapse reduction, major efforts focused on reducing acute and late toxicity through advanced risk adaptation of treatment. The major findings derived from these ALL-BFM trials were as follows: 1) preventive cranial radiotherapy could be safely reduced to 12 Gy in T-ALL and high-risk ALL patients and eliminated in non-high-risk non-T-ALL patients, if it was replaced by high-dose and intrathecal methotrexate; 2) omission of delayed reintensification severely impaired outcome of low-risk patients; 3) 6 months less maintenance therapy caused an increase in systemic relapses; 4) slow response to an initial 7-day prednisone window was identified as adverse prognostic factor; 5) condensed induction therapy resulted in a significant improvement of outcome; 6) the daunorubicin dose in induction could be safely reduced in low-risk patients; 7) intensification of consolidation/reintensification treatment led to considerable improvement of outcome in high-risk patients.

Involvement of the CD95 (APO-1/FAS) receptor/ligand system in drug-induced apoptosis in leukemia cells.

Cytotoxic drugs used in chemotherapy of leukemias and solid tumors cause apoptosis in target cells. In lymphoid cells the CD95 (APO-1/Fas)/CD95 ligand (CD95-L) system is a key regulator of apoptosis. Here we describe that doxorbicin induces apoptosis via the CD95/CD95-L system in human leukemia T-cell lines. Doxorubicin-induced apoptosis was completely blocked by inhibition of gene expression and protein synthesis. Also, doxorbicin strongly stimulates CD95-L messenger RNA expression in vitro at concentrations relevant for therapy in vivo. CEM and jurkat cells resistant to CD95-mediated apoptosis were also resistant to doxorbicin-induced apoptosis . Furthermore, doxorbicin-induced apoptosis was inhibited by blocking F(ab')2 anti-APO-1 (anti-CD95) antibody fragments. Expression of CD95-L mRNA and protein in vitro was also stimulated by other cytotoxic drugs such as methotrexate. The finding that apoptosis caused by anticancer drugs may be mediated via the CD95 system provides a new molecular insight into resistance and sensitivity toward chemotherapy in malignancies.

Utilization of psychosocial care and oncological follow-up assessments among German long-term survivors of cancer with onset during adolescence.

BACKGROUND: Survivors of pediatric cancer are at increased risk for medical and psychosocial late effects. This study retrospectively investigated the utilization of oncological and psychosocial care by former adolescent cancer patients (≥ 5 years since cancer diagnosis) in Germany. PATIENTS: Based on data of the German Childhood Cancer Registry (N=1 876 survivors of cancer with an age at diagnosis between 15 and 18 years), the study cohort comprised 820 survivors of adolescent cancer (time since diagnosis: M=13.7, SD=6.0, age at follow-up: M=30.4, SD=6.0 years). METHOD: Survivors of adolescent cancer completed standardized questionnaires measuring symptoms of posttraumatic stress, depression and anxiety as well as items on their utilization of medical and psychosocial care. RESULTS: More than a quarter (26.2%) of the survivors was no longer attending regular oncological follow-up assessments. Less than half of the survivors (44.4%) had received psychosocial care, mostly during their in-patient cancer treatment and their post-acute rehabilitation phase. Out of 184 survivors showing clinically relevant symptoms of posttraumatic stress, anxiety and/or depression at time of the study, 12.0% received psychosocial care and 13.6% took psychotropic medication. CONCLUSION: It should be studied further why only a small proportion of the survivors showing clinically relevant symptoms received psychosocial or psychopharmacological treatment. Systematic oncological follow-up assessments should take psychological late effects into greater account.

Hereditary hyperferritinemia cataract syndrome: clinical, genetic, and laboratory findings in 5 families.

BACKGROUND: The hereditary hyperferritinemia cataract syndrome (HHCS) is an autosomal dominant disorder characterized by high serum ferritin and early onset cataract. Mutations in the iron responsive element (IRE) within the 5' untranslated region of the L-ferritin (FTL) gene lead to constitutive L-ferritin synthesis resulting in hyperferritinemia. Bilateral cataract formation is caused by the intracellular accumulation of ferritin in the lens. PATIENTS: 4 children from unrelated families were referred for further exploration of hyperferritinemia which was detected during the diagnostic work-up of gastroenterological or hematological disorders. 1 patient was primarily referred for the investigation of bilateral cataract.Diagnostics included routine blood analysis, including complete blood count, iron status, liver and kidney parameters, a physical and an ophthalmological examination. Molecular genetic analysis of the FTL IRE was performed in 4 patients by PCR from genomic DNA and subsequent direct sequencing. RESULTS: All index patients presented with isolated hyperferritinemia without iron overload and had a positive family history for early onset cataract. Age at onset and disease severity varied between different families and among family members. Molecular genetic analysis revealed point mutations within the FTL IRE. CONCLUSION: In patients with hyperferritinemia but without any other sign of iron overload or inflammation HHCS should be considered to avoid complex and invasive procedures. Vice versa, in patients with familial inherited cataract the early serum ferritin measurement helps to avoid unnecessary diagnostics.

Latent TGFß-binding proteins regulate UCP1 expression and function via TGFß2.

OBJECTIVE: Activation of brown adipose tissue (BAT) in humans has been proposed as a new treatment approach for combating obesity and its associated diseases, as BAT participates in the regulation of energy homeostasis as well as glucose and lipid metabolism. Genetic contributors driving brown adipogenesis in humans have not been fully understood. METHODS: Profiling the gene expression of progenitor cells from subcutaneous and deep neck adipose tissue, we discovered new secreted factors with potential regulatory roles in white and brown adipogenesis. Among these, members of the latent transforming growth factor beta-binding protein (LTBP) family were highly expressed in brown compared to white adipocyte progenitor cells, suggesting that these proteins are capable of promoting brown adipogenesis. To investigate this potential, we used CRISPR/Cas9 to generate LTBP-deficient human preadipocytes. RESULTS: We demonstrate that LTBP2 and LTBP3 deficiency does not affect adipogenic differentiation, but diminishes UCP1 expression and function in the obtained mature adipocytes. We further show that these effects are dependent on TGFß2 but not TGFß1 signaling: TGFß2 deficiency decreases adipocyte UCP1 expression, whereas TGFß2 treatment increases it. The activity of the LTBP3-TGFß2 axis that we delineate herein also significantly correlates with UCP1 expression in human white adipose tissue (WAT), suggesting an important role in regulating WAT browning as well. CONCLUSIONS: These results provide evidence that LTBP3, via TGFß2, plays an important role in promoting brown adipogenesis by modulating UCP1 expression and mitochondrial oxygen consumption.

[Spontaneously occurring pneumomediastinum related to a pneumopericardium, a pneumothorax and a skin emphysema in a 12-year old boy]. / Spontan aufgetretenes Pneumomediastinum, assoziiert mit Pneumoperikard, Pneumothorax und Hautemphysem bei einem 12-jährigen Jungen.

BACKGROUND: The spontaneous pneumomediastinum describes the existence of air inside the mediastinum, is caused by an alveolar rupture in most cases and occurs without influence of traumata, surgery or iatrogen interferences. This is a rare and, in most cases, benign disease. An association between a pneumomediastinum, a pneumopericardium, a pneumothorax, as well as a skin emphysema, is extremely rare in children and young adults. CASUISTICS: After doing sports a 12-year-old boy with a bronchial asthma history complained about acute thoracic pain and dyspnea. Later on, the boy noticed a breathing sound based on his breathing cycle and skin emphysema at the left side of his thorax and neck, which was not painful. Based on the anamnesis, the clinical examination, the results of different diagnostic methods, including a thoracic X-ray in 2 levels, the boy was diagnosed with an acute spontaneous pneumomediastinum in association with a pneumopericardium, a pneumothorax and a skin emphysema. After diagnostic validation and exclusion of different diagnoses as well as an osseous chest trauma, the 12-year old patient was treated symptomatically. A complete absorption of the different air leaks could be described. CONCLUSIONS: In this case report and literature review possible causes, differential diagnoses, and treatments are summarized. Based on this, we suggest that this case report serve as a diagnostic guide for patients presenting with these symptoms.

4-hydroperoxy-cyclophosphamide mediates caspase-independent T-cell apoptosis involving oxidative stress-induced nuclear relocation of mitochondrial apoptogenic factors AIF and EndoG.

Apoptosis is a major mechanism of treatment-induced T-cell depletion in leukemia and autoimmune diseases. While 'classical' apoptosis is considered to depend on caspase activation, caspase-independent death is increasingly recognized as an alternative pathway. Although the DNA-damaging drug cyclophosphamide (CY) is widely used for therapy of hematological malignancies and autoimmune disorders, the molecular mechanism of apoptosis induction remains largely unknown. Here, we report that treatment of Jurkat, cytotoxic, and primary leukemic T cells with an activated analog of CY, 4-hydroperoxy-cyclophosphamide (4-OOH-CY), induces caspase activation and typical features of apoptosis, although cell death was not prevented by caspase inhibition. Also depletion of murine thymocytes and splenocytes after CY treatment in vivo was not inhibited by Z-Val-Ala-DL-Asp-fluoromethylketone (Z-VAD.fmk). Caspase-8 and receptor-induced protein (RIP) were dispensable for 4-OOH-CY-mediated apoptosis, while overexpression of Bcl-2 was partially protective. 4-OOH-CY treatment induced reactive oxygen species production, upregulation of Bax, and nuclear relocation of the mitochondrial factors apoptosis-inducing factor (AIF) and endonuclease G (EndoG). The antioxidant N-acetyl-L-cysteine substantially inhibited conformational changes of Bax, loss of mitochondrial membrane potential, nuclear relocation of mitochondrial factors, and apoptosis induction in 4-OOH-CY-treated T cells. These results strongly indicate that oxidative damage-induced nuclear translocation of AIF and EndoG in 4-OOH-CY-treated T cells might represent an alternative death pathway in the absence of caspase activity.

Hematopoietic stem cell transplantation from matched unrelated donors in chronic granulomatous disease.

We report on 12 patients with chronic granulomatous disease transplanted with hematopoietic stem cells from matched unrelated (n = 9) or matched sibling donors (n = 3). The most common infectious complication was pulmonary aspergillosis, which nine patients had previously developed. Only 5 of 12 individuals had normal lung function prior to transplantation. At a mean follow-up of 53 months 9 of the 12 patients are alive including 7 of 9 following matched unrelated donor (MUD) transplantation. One patient died from ARDS, another from systemic BK virus infection, the third from complications of chronic graft-versus-host disease. Seven of nine surviving patients have normal lung function now. HSCT from a MUD is an option worth considering when no matched family donor is available. Restricted lung function prior to HSCT does not appear to be a limiting factor for such treatment.

Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity.

Fas (also known as Apo1 and CD95) is a cell surface receptor involved in apoptotic cell death. Fas expression and function were analyzed in three children (including two siblings) with a lymphoproliferative syndrome, two of whom also had autoimmune disorders. A large deletion in the gene encoding Fas and no detectable cell surface expression characterized the most affected patient. Clinical manifestations in the two related patients were less severe: Fas-mediated apoptosis was impaired and a deletion within the intracytoplasmic domain was detected. These findings illustrate the crucial regulatory role of Fas and may provide a molecular basis for some autoimmune diseases in humans.

Monoclonal antibody-mediated tumor regression by induction of apoptosis.

To characterize cell surface molecules involved in control of growth of malignant lymphocytes, monoclonal antibodies were raised against the human B lymphoblast cell line SKW6.4. One monoclonal antibody, anti-APO-1, reacted with a 52-kilodalton antigen (APO-1) on a set of activated human lymphocytes, on malignant human lymphocyte lines, and on some patient-derived leukemic cells. Nanogram quantities of anti-APO-1 completely blocked proliferation of cells bearing APO-1 in vitro in a manner characteristic of a process called programmed cell death or apoptosis. Cell death was preceded by changes in cell morphology and fragmentation of DNA. This process was distinct from antibody- and complement-dependent cell lysis and was mediated by the antibody alone. A single intravenous injection of anti-APO-1 into nu/nu mice carrying a xenotransplant of a human B cell tumor induced regression of this tumor within a few days. Histological thin sections of the regressing tumor showed that anti-APO-1 was able to induce apoptosis in vivo. Thus, induction of apoptosis as a consequence of a signal mediated through cell surface molecules like APO-1 may be a useful therapeutic approach in treatment of malignancy.

Growth control of normal and malignant lymphocytes--cell death research from basic concepts to signal pathways and translation into the clinic.

Twenty years ago the fist bona fide death receptor, APO-1/FAS/CD95 was discovered along with the pathways that regulate programmed cell death or apoptosis. From the very beginning, this research was considered to have substantial impact on diseases and to provide a rational strategy for therapeutic intervention. In particular cell death research proved to be the key for the development of novel strategies for cancer therapy. In the past two decades, deregulated apoptosis in tumors has been delineated and possible targets for therapeutic intervention have been identified. However, it still took a long way until this work could be translated into clinical trials only in the past few years. Current strategies involve modification of apoptosis signalling based on our knowledge of sensitivity and resistance for apoptosis induction rather than the use of individual agents for cytotoxicity. In this review, an overview of the developments in the field from basic discoveries to the recent clinical trials is given.

Coincidence of recurrent hemiparesis and detection of ALL in a 4-year-old girl: one or two diseases?

Stroke like symptoms in children such as hemiparesis are often associated with infection, cranial trauma, cardiac anomalies or sickle cell disease. In childhood leukemia, stroke like symptoms at presentation are rare and normally caused by cerebral bleedings. Here we report a patient who presented with classical stroke symptoms and hemiparesis prior to the diagnosis of acute lymphoblastic leukemia without proven CNS infiltration by leukemic cells. In general, acute leukemia or cerebral lymphoma do not lead to extensive defects of brain tissue. This unusual case suggests that acute lymphoblastic leukemia may present with stroke like CNS symptoms including hemiparesis.

Elevated UCP1 levels are sufficient to improve glucose uptake in human white adipocytes.

Brown adipose tissue (BAT) has been considered beneficial for metabolic health by participating in the regulation of glucose homoeostasis. The browning factors that improve glucose uptake beyond normal levels are still unknown but glucose uptake is not affected in UCP1 knockout mice. Here, we demonstrate in human white adipocytes that basal/resting glucose uptake is improved by solely elevating UCP1 protein levels. Generating human white Simpson-Golabi-Behmel syndrome (SGBS) adipocytes with a stable knockout and overexpression of UCP1, we discovered that UCP1 overexpressing adipocytes significantly improve glucose uptake by 40%. Mechanistically, this is caused by higher glycolytic flux, seen as increased oxygen consumption, extracellular acidification and lactate secretion rates. The improvements in glucose handling are comparable to white-to-brown transitions, as judged by, for the first time, directly comparing in vitro differentiated mouse brown vs white adipocytes. Although no adipogenic, metabolic and mitochondrial gene expressions were significantly altered in SGBS cells, pharmacological inhibition of GLUT1 completely abrogated differences between UCP1+ and control cells, thereby uncovering GLUT1-mediated uptake as permissive gatekeeper. Collectively, our data demonstrate that elevating UCP1 levels is sufficient to improve human white adipocytes as a glucose sink without adverse cellular effects, thus not requiring the adrenergic controlled, complex network of browning which usually hampers translational efforts.

Role of hypoxia inducible factor-1 alpha in modulation of apoptosis resistance.

Hypoxia inducible factor-1 (HIF-1) is the major transcription factor and key regulator of adoptive responses to hypoxia. Although it usually promotes tumor cell survival under hypoxia, it has also been implied to trigger apoptosis. Although the impact of hypoxia has been extensively studied in many adult solid tumors, its role in most childhood tumors, for example, in rhabdomyosarcoma (RMS) or Ewing sarcoma (ES), has not yet been addressed. Here, we report that hypoxia protects A204 RMS and A673 ES cells against anticancer drug- or tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis and that Hif-1alpha plays a key role in conferring apoptosis resistance under hypoxia. Although a functional HIF-1 pathway and proapoptotic proteins such as p53 and Bcl-2/E1B 19 kDa interacting protein 3 were activated under hypoxia in both A204 RMS and A673 ES cells, these cells remained refractory to apoptosis. Concomitant analysis of antiapoptotic proteins revealed that hypoxia induced expression of Bcl-2 and inhibitor of apoptosis proteins (IAP)-2 as well as proteins associated with anaerobic metabolism such as the glucose transporter protein GLUT-1 and the glycolytic enzyme Aldolase A. Specific downregulation of Hif-1alpha by RNA interference significantly enhanced apoptosis under hypoxia by preventing the hypoxia-mediated increase in GLUT-1 expression without altering expression levels of the antiapoptotic proteins Bcl-2 or cIAP-2. Moreover, glucose deprivation-induced apoptosis of A204 RMS and A673 ES cells was inhibited under hypoxic conditions in a Hif-1alpha-dependent manner. As GLUT-1 was induced via Hif-1alpha under hypoxia in A204 RMS and A673 ES, these findings suggest that the Hif-1alpha-mediated increase in glucose uptake plays an important role in conferring apoptosis resistance. Thus, hypoxia-inducible genes may represent novel targets for therapeutic intervention in some pediatric tumors, which warrants further investigation.