Lentivirus-mediated gene therapy corrects ribosomal biogenesis and shows promise for Diamond Blackfan anemia.

This study lays the groundwork for future lentivirus-mediated gene therapy in patients with Diamond Blackfan anemia (DBA) caused by mutations in ribosomal protein S19 (RPS19), showing evidence of a new safe and effective therapy. The data show that, unlike patients with Fanconi anemia (FA), the hematopoietic stem cell (HSC) reservoir of patients with DBA was not significantly reduced, suggesting that collection of these cells should not constitute a remarkable restriction for DBA gene therapy. Subsequently, 2 clinically applicable lentiviral vectors were developed. In the former lentiviral vector, PGK.CoRPS19 LV, a codon-optimized version of RPS19 was driven by the phosphoglycerate kinase promoter (PGK) already used in different gene therapy trials, including FA gene therapy. In the latter one, EF1α.CoRPS19 LV, RPS19 expression was driven by the elongation factor alpha short promoter, EF1α(s). Preclinical experiments showed that transduction of DBA patient CD34+ cells with the PGK.CoRPS19 LV restored erythroid differentiation, and demonstrated the long-term repopulating properties of corrected DBA CD34+ cells, providing evidence of improved erythroid maturation. Concomitantly, long-term restoration of ribosomal biogenesis was verified using a potentially novel method applicable to patients' blood cells, based on ribosomal RNA methylation analyses. Finally, in vivo safety studies and proviral insertion site analyses showed that lentivirus-mediated gene therapy was nontoxic.

Diagnosis, treatment, and surveillance of Diamond-Blackfan anaemia syndrome: international consensus statement.

Diamond-Blackfan anaemia (DBA), first described over 80 years ago, is a congenital disorder of erythropoiesis with a predilection for birth defects and cancer. Despite scientific advances, this chronic, debilitating, and life-limiting disorder continues to cause a substantial physical, psychological, and financial toll on patients and their families. The highly complex medical needs of affected patients require specialised expertise and multidisciplinary care. However, gaps remain in effectively bridging scientific discoveries to clinical practice and disseminating the latest knowledge and best practices to providers. Following the publication of the first international consensus in 2008, advances in our understanding of the genetics, natural history, and clinical management of DBA have strongly supported the need for new consensus recommendations. In 2014 in Freiburg, Germany, a panel of 53 experts including clinicians, diagnosticians, and researchers from 27 countries convened. With support from patient advocates, the panel met repeatedly over subsequent years, engaging in ongoing discussions. These meetings led to the development of new consensus recommendations in 2024, replacing the previous guidelines. To account for the diverse phenotypes including presentation without anaemia, the panel agreed to adopt the term DBA syndrome. We propose new simplified diagnostic criteria, describe the genetics of DBA syndrome and its phenocopies, and introduce major changes in therapeutic standards. These changes include lowering the prednisone maintenance dose to maximum 0·3 mg/kg per day, raising the pre-transfusion haemoglobin to 9-10 g/dL independent of age, recommending early aggressive chelation, broadening indications for haematopoietic stem-cell transplantation, and recommending systematic clinical surveillance including early colorectal cancer screening. In summary, the current practice guidelines standardise the diagnostics, treatment, and long-term surveillance of patients with DBA syndrome of all ages worldwide.

Bone marrow disease in rhabdomyosarcoma visualized by 2-[18F]fluorodeoxyglucose positron emission tomography/computed tomography.

Bone marrow metastases-noted in 6% of patients with rhabdomyosarcoma-have been linked to very poor outcomes. Bilateral bone marrow sampling from iliac crests has been the gold standard for bone marrow examination in rhabdomyosarcoma, but sampling errors due to patchy bone marrow involvement may limit its sensitivity. Here, we report the case of a 6-year-old boy with embryonal rhabdomyosarcoma of the skull base and multiple 2-[18F]fluoro-2-deoxy-D-glucose (2-[18F]FDG)-avid bone marrow metastases visualized by positron emission tomography and computed tomography (2-[18F]FDG PET/CT). His bone marrow aspirates were tumor-free. This case illustrates the diagnostic value of 2-[18F]FDG PET/CT in the detection of bone marrow metastases in rhabdomyosarcoma patients, which may re-shape the definition of bone marrow disease and, ultimately, alter disease staging and risk stratification.

A Multimodal Lifestyle Psychosocial Survivorship Program in Young Cancer Survivors: The CARE for CAYA Program-A Randomized Clinical Trial Embedded in a Longitudinal Cohort Study.

Importance: There is a lack of trials examining the effect of counseling interventions for child, adolescent, and younger adult (CAYA) cancer survivors. Objective: To assess lifestyle habits and the psychosocial situation of CAYAs to determine the efficacy of needs-based interventions in the CARE for CAYA program (CFC-P). Design, Setting, and Participants: The CFC-P was conducted as a multicenter program in 14 German outpatient clinics, mainly university cancer centers. Recruitment began January 1, 2018; a randomized clinical trial was conducted until July 15, 2019; and intervention was continued as a longitudinal cohort study until March 31, 2021. Data preparation was conducted from April 1, 2021, and analysis was conducted from August 14, 2021, to May 31, 2022. Herein, predefined confirmatory analyses pertain to the RCT and descriptive results relate to the overall longitudinal study. Data analysis was based on the full analysis set, which is as close as possible to the intention-to-treat principle. Intervention: A comprehensive assessment determined needs in physical activity, nutrition and psychooncology. Those with high needs participated in 1 to 3 modules. In the RCT, the IG received 5 counseling sessions plus newsletters, while the control group CG received 1 counseling session. Main Outcomes and Measures: The primary outcome was the change in the rate of CAYAs with high needs at 52 weeks. Secondary outcomes were feasibility, modular-specific end points, satisfaction, quality of life, and fatigue. Results: Of 1502 approached CAYAs aged 15 to 39 years, 692 declined participation. Another 22 CAYAs were excluded, resulting in 788 participants. In the randomized clinical trial, 359 CAYAs were randomized (intervention group [IG], n = 183; control group [CG], n = 176), and 274 were followed up. In the RCT, the median age was 25.0 (IQR, 19.9-32.2) years; 226 were female (63.0%) and 133 male (37.0%). After 52 weeks, 120 CAYAs (87.0%) in the IG and 115 (86.5%) in the CG still had a high need in at least 1 module (odds ratio, 1.04; 95% CI, 0.51-2.11; P = .91). Both groups reported reduced needs, improved quality of life, reduced fatigue, and high satisfaction with the CFC-P. Conclusions and Relevance: In this randomized clinical trial, the implementation of a lifestyle program in this cohort was deemed necessary, despite not meeting the primary outcome. The interventions did not alter the rate of high needs. The results may provide guidance for the development of multimodal interventions in the follow-up care of CAYAs. Trial Registration: German Clinical Trial Register: DRKS00012504.

Diagnostic evaluation of paediatric autoimmune lymphoproliferative immunodeficiencies (ALPID): a prospective cohort study.

BACKGROUND: Lymphoproliferation and autoimmune cytopenias characterise autoimmune lymphoproliferative syndrome. Other conditions sharing these manifestations have been termed autoimmune lymphoproliferative syndrome-like diseases, although they are frequently more severe. The aim of this study was to define the genetic, clinical, and immunological features of these disorders to improve their diagnostic classification. METHODS: In this prospective cohort study, patients were referred to the Center for Chronic Immunodeficiency in Freiburg, Germany, between Jan 1, 2008 and March 5, 2022. We enrolled patients younger than 18 years with lymphoproliferation and autoimmune cytopenia, lymphoproliferation and at least one additional sign of an inborn error of immunity (SoIEI), bilineage autoimmune cytopenia, or autoimmune cytopenia and at least one additional SoIEI. Autoimmune lymphoproliferative syndrome biomarkers were determined in all patients. Sanger sequencing followed by in-depth genetic studies were recommended for patients with biomarkers indicative of autoimmune lymphoproliferative syndrome, while IEI panels, exome sequencing, or genome sequencing were recommended for patients without such biomarkers. Genetic analyses were done as decided by the treating physician. The study was registered on the German Clinical Trials Register, DRKS00011383, and is ongoing. FINDINGS: We recruited 431 children referred for autoimmune lymphoproliferative syndrome evaluation, of whom 236 (55%) were included on the basis of lymphoproliferation and autoimmune cytopenia, 148 (34%) on the basis of lymphoproliferation and another SoIEI, 33 (8%) on the basis of autoimmune bicytopenia, and 14 (3%) on the basis of autoimmune cytopenia and another SoIEI. Median age at diagnostic evaluation was 9·8 years (IQR 5·5-13·8), and the cohort comprised 279 (65%) boys and 152 (35%) girls. After biomarker and genetic assessments, autoimmune lymphoproliferative syndrome was diagnosed in 71 (16%) patients. Among the remaining 360 patients, 54 (15%) had mostly autosomal-dominant autoimmune lymphoproliferative immunodeficiencies (AD-ALPID), most commonly affecting JAK-STAT (26 patients), CTLA4-LRBA (14), PI3K (six), RAS (five), or NFκB (three) signalling. 19 (5%) patients had other IEIs, 17 (5%) had non-IEI diagnoses, 79 (22%) were unresolved despite extended genetics (ALPID-U), and 191 (53%) had insufficient genetic workup for diagnosis. 16 (10%) of 161 patients with a final diagnosis had somatic mutations. Alternative classification of patients fulfilling common variable immunodeficiency or Evans syndrome criteria did not increase the proportion of genetic diagnoses. INTERPRETATION: The ALPID phenotype defined in this study is enriched for patients with genetic diseases treatable with targeted therapies. The term ALPID might be useful to focus diagnostic and therapeutic efforts by triggering extended genetic analysis and consideration of targeted therapies, including in some children currently classified as having common variable immunodeficiency or Evans syndrome. FUNDING: Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy. TRANSLATION: For the German translation of the abstract see Supplementary Materials section.

Current German Guidelines on Diagnosis and Treatment of Secondary Hemochromatosis in Patients with Congenital Anemias.

Adequate diagnosis and treatment of secondary hemochromatosis in patients with congenital anemias is important for reducing long-term mortality and morbidity as for improving patients' quality of life. Due to the strong migration movements during recent years, the number of patients in Germany suffering from hemoglobinopathies as some of the most relevant disorders in the context of iron overload (IOL) has increased enormously. Many of these patients had received inadequate medical care in their countries of origin prior to migration, including diagnosis and treatment of IOL. In parallel, various medical developments and achievements took place, including the expansion of stem cell transplant as curative therapeutic option and the introduction of gene therapy for patients with hemoglobinopathies. Diagnostic tools to assess both liver and heart IOL became available at more sites in Germany. Overall experience with iron elimination therapy either as monotherapy or as combination of different chelators increased. All these aspects were considered during revision of the consensus-based guideline on the diagnosis and treatment of secondary IOL in patients with congenital anemias (AWMF Reg. No. 025/029). Here, we briefly summarize the procedure for the revision of the guideline, provide a brief overview of innovations as compared to the previous version dated from 2015, and finally present the consensus recommendations adopted in the current version.

Multimodal Treatment of Nasopharyngeal Carcinoma in Children, Adolescents and Young Adults-Extended Follow-Up of the NPC-2003-GPOH Study Cohort and Patients of the Interim Cohort.

Nasopharyngeal carcinoma (NPC) in children and young adults has been treated within two consecutive prospective trials in Germany, the NPC-91 and the NPC-2003 study of the German Society of Pediatric Oncology and Hematology (GPOH). In these studies, multimodal treatment with induction chemotherapy, followed by radio (chemo)therapy and interferon-beta maintenance, yielded promising survival rates even after adapting total radiation doses to tumor response. The outcome of 45 patients in the NPC-2003 study was reassessed after a median follow-up of 85 months. In addition, we analyzed 21 further patients after closure of the NPC-2003 study, recruited between 2011 and 2017, and treated as per the NPC-2003 study protocol. The EFS and OS of 66 patients with locoregionally advanced NPC were 93.6% and 96.7%, respectively, after a median follow-up of 73 months. Seven patients with CR after induction therapy received a reduced radiation dose of 54 Gy; none relapsed. In young patients with advanced locoregional NPC, excellent long-term survival rates can be achieved by multimodal treatment, including interferon-beta. Radiation doses may be reduced in patients with complete remission after induction chemotherapy and may limit radiogenic late effects.

Unusual course of disease and genetic profile in Li-Fraumeni syndrome-associated osteosarcoma - a case report.

BACKGROUND: Osteosarcoma is a highly malignant tumour associated with numerous and complex genetic alterations like copy number alterations. Recent whole genome studies revealed distinct mutations in several candidate oncogenes. While clinical parameters stratify osteosarcoma patients in risk groups, genetic profiles have not yet been used to tailor tumour treatment. However, specific copy number alterations seem to have a prognostic impact in osteosarcoma treatment. Somatic TP53 gene mutation frequently occurs in sporadic osteosarcoma. When arising germline, TP53 mutation leads to Li-Fraumeni syndrome and may result in early life osteosarcoma. The effect of Li-Fraumeni syndrome on the genetic profile of osteosarcoma and the consideration of the syndrome during cancer treatment are topics of current research. CASE PRESENTATION: We report a 25-year-old female with pelvic osteosarcoma refusing continuation of therapy. She interrupted neo-adjuvant chemotherapy according to EURAMOS-1/COSS recommendations and declined local or further adjuvant therapy. Surprisingly, she remained in sustained remission for the osteosarcoma but eventually died from newly diagnosed breast cancer. After establishment of breast cancer, we detected TP53 germline mutation and investigated the osteosarcoma material with array-CGH. CONCLUSION: Genetic examination of the tumour evidenced several copy number alterations with striking differences to previously reported data. We discuss possible influences of the genetic profile on the unusual clinical course and the significance of Li-Fraumeni syndrome for the genetic profile. Specific loss of (proto-) oncogenes might have contributed to the unusual case. Further large-scale genetics of Li-Fraumeni patients combined with detailed clinical data will help to identify specific genetic risk profiles and improve treatment.

Hemangioblastoma and von Hippel-Lindau disease: genetic background, spectrum of disease, and neurosurgical treatment.

INTRODUCTION: Hemangioblastomas are rare, histologically benign, highly vascularized tumors of the brain, the spinal cord, and the retina, occurring sporadically or associated with the autosomal dominant inherited von Hippel-Lindau (VHL) disease. Children or adults with VHL disease have one of > 300 known germline mutations of the VHL gene located on chromosome 3. They are prone to develop hemangioblastomas, extremely rarely starting at age 6, rarely at age 12-18, and, typically and almost all, as adults. There is a plethora of VHL-associated tumors and cysts, mainly in the kidney, pancreas, adrenals, reproductive organs, and central nervous system. Due to a lack of causal treatment, alleviation of symptoms and prevention of permanent neurological deficits as well as malignant transformation are the main task. Paucity of data and the nonlinear course of tumor progression make management of pediatric VHL patients with hemangioblastomas challenging. METHODS: The Freiburg surveillance protocol was developed by combining data from the literature and our experience of examinations of > 300 VHL patients per year at our university VHL center. RESULTS: Key recommendations are to start screening of patients at risk by funduscopy with dilated pupils for retinal tumors with admission to school and with MRI of the brain and spinal cord at age 14, then continue biannually until age 18, with emergency MRI in case of neurological symptoms. Indication for surgery remains personalized and should be approved by an experienced VHL board, but we regard neurological symptoms, rapid tumor growth, or critically large tumor/cyst sizes as the key indications to remove hemangioblastomas. Since repeated surgery on hemangioblastomas in VHL patients is not rare, modern neurosurgical techniques should encompass microsurgery, neuronavigation, intraoperative neuromonitoring, fluorescein dye-based intraoperative angiography, intraoperative ultrasound, and minimally invasive approaches, preceded in selected cases by endovascular embolization. Highly specialized neurosurgeons are able to achieve a very low risk of permanent morbidity for the removal of hemangioblastomas from the cerebellum and spinal cord. Small retinal tumors of the peripheral retina can be treated by laser coagulation, larger tumors by cryocoagulation or brachytherapy. CONCLUSION: We consider management at experienced VHL centers mandatory and careful surveillance and monitoring of asymptomatic lesions are required to prevent unnecessary operations and minimize morbidity.

Abscessing Infection by Streptococcus mitis Mimicking Metastatic Lesions in a 5-Year-Old Girl With Nephroblastoma: A Case Report.

Streptococcus mitis is a common pathogen causing infections in oncological patients. However, cases of abscesses caused by Streptococcus mitis in oncological patients have not been reported so far. We report on 5-year-old child with nephroblastoma and pulmonary and hepatic metastases at diagnosis who went into complete remission undergoing chemotherapy and nephrectomy, and who developed new round lesions in liver and lungs under continuous chemotherapy suggestive of new metastases. Biopsy of the lesions revealed abscesses with detection of Streptococcus mitis. The child was successfully treated with antibiotics, finished chemotherapy per protocol and has been in complete remission for 14 months. Infectious lesions involving organs of typical metastatic dissemination can easily be misdiagnosed as metastases, especially in the absence of symptoms. Histologic proof of lesions suspicious of metastases is mandatory if it leads to a change of prognosis and therapy. Streptococcus mitis can be a causative organism of pulmonary and hepatic abscesses in oncological patients.

Lethal course of meconium ileus in preterm twins revealing a novel cystic fibrosis mutation (p.Cys524Tyr).

BACKGROUND: In term newborns meconium ileus is frequently associated with cystic fibrosis. Reports on meconium ileus in preterm infants being diagnosed with cystic fibrosis early after birth are very scarce. Associations between genotype and phenotype in cystic fibrosis and its particular comorbidities have been reported. CASE PRESENTATION: Two extremely preterm twin infants (26 weeks of gestation) born from a Malaysian mother and a Caucasian father were presented with typical signs of meconium ileus. Despite immediate surgery both displayed a unique and finally lethal course. Mutation analysis revealed a novel, probably pathogenic cystic fibrosis mutation, p.Cys524Tyr. The novel mutation might explain the severity of disease next to typical sequelae of prematurity. CONCLUSION: Preterm neonates with meconium ileus have to be evaluated for cystic fibrosis beyond ethnical boundaries, but may take devastating clinical courses despite early treatment. The novel, potentially pathogenic CF mutation p.Cys524Tyr might be associated with severe meconium ileus in neonates. Disease-modifying loci are important targets for intestinal comorbidity of cystic fibrosis.

Thalidomide has anti-inflammatory properties in neonatal immune cells.

Neonates demonstrate functional immaturity and dysregulation of immune responses leading to systemic inflammation and enhanced apoptosis of immune cells. Thalidomide has already been proven to differentially regulate immune responses and support anti-apoptosis in immunodeficiency syndromes. Thus, it was the aim of this study to evaluate the effects of thalidomide on the cytokine response and apoptosis of neonatal immune cells. After whole blood culture and stimulation of cord and adult blood samples, the intracytoplasmic expression and the secreted amounts of IL-2, TNF-α, IFN-γ, IL-6, IL-10 and IL-8 were assessed by flow cytometry and Cytokine Bead Array. Apoptosis was detected using Annexin-V staining. Bcl-2 expression was analysed using the Cytokine Bead Array Apoptosis Kit. Exposure to thalidomide (100 µg/ml) reduced the intracytoplasmic pro-inflammatory cytokine production of neonatal monocytes and the IFN-γ production of neonatal lymphocytes. In supernatants, the addition of thalidomide resulted in reduction of TNF-α, IL-6, IL-10 and, by trend, IFN-γ. While stimulated neonatal lymphocytes exhibited susceptibility to apoptosis, thalidomide tended to diminish apoptotic cells. Bcl-2 expression tended to be increased after addition of thalidomide. The potent anti-inflammatory effects of thalidomide and its anti-apoptotic properties in cord blood immune cells provide the basis for future strategies to optimise treatment of neonatal infections and immunodeficiency syndromes.

Insulin-like growth factor-I regulates the neonatal immune response in infection and maturation by suppression of IFN-γ.

Disturbances in Insulin-like growth factor-I (IGF-I) and dysregulation in neonatal immune responses have been associated with typical neonatal diseases. Immunosuppression by IGF-I might be a key regulator of neonatal immune responses in infection and maturation. However, information on IGF-I serum levels, IGF-I-receptor (IGF-IR) and effects on functional properties of neonatal immune cells is scarce. Neonatal cord blood samples were stimulated with anti-CD3/anti-CD28, PMA/ionomycin or LPS in a whole-blood assay, while IGF-I serum levels and IGF-I receptor expression were assessed. Furthermore the effect of IGF-I on cytokine expression, apoptosis and the DNA binding activity of AP-1 and NFκB was evaluated. IGF-I serum levels were within normal range. The IGF-I-receptor was present on the surface of cord blood CD3+CD4+lymphocytes and cord blood CD14+monocytes. Upon stimulation the IGF-I-R expression on lymphocytes was significantly upregulated. The addition of IGF-I (100ng/ml) to whole blood cultures inhibited the secretion of IFN-γ from PMA/ionomycin-stimulated cord blood mononuclear cells (CBMC). While IGF-I did not influence apoptosis in our experimental setting, it led to a distinct decrease in anti-CD3/CD28-stimulated DNA binding activity of AP-1 and NFκB. Thus IGF-I may be a key regulator of neonatal immune responses in maturation processes and inflammation by suppressing proinflammatory Th1 responses. Future in vivo studies need to elucidate whether disturbances of the IGF-I serum level and IGF-IR expression are associated with susceptibility for infections and subsequent diseases in neonates.

Plasmacytoid dendritic cell subpopulations in head and neck squamous cell carcinoma.

Human plasmacytoid dendritic cells (PDCs) are present in solid tumor tissue and metastatic cervical lymph nodes (CLN) in head and neck squamous cell carcinoma (HNSCC). We recently showed that classical PDC functions are heavily disturbed in the tumor microenvironment. In this study we present a new approach to the subject by introducing 3 PDC subsets in HNSCC, characterized by the surface markers CD25, CD56 and CD203c. The first subset, positive for CD25, is significantly induced by HNSCC in vitro and present in metastatic lymph nodes in vivo. This subset can be phenotypically subdivided into matured cells and into a group expressing early T cell markers. Functionally this subgroup is associated with the secretion of IL-8. The second subset, positive for CD56, constitutes 4-5% of all PDCs and is significantly down-regulated by HNSCC. Furthermore, this population sporadically expresses perforin/granzyme B and is absent in metastatic lymph nodes. The third subset, positive for the basophile marker CD203c, is inducible by crosslinking BDCA-2 in the presence of HNSCC and IL-4. Future studies will have to clarify the in vivo relevance of the different PDC subsets in HNSCC.

Expression of the T cell receptor αß on a CD123+ BDCA2+ HLA-DR+ subpopulation in head and neck squamous cell carcinoma.

Human Plasmacytoid Dendritic Cells (PDCs) infiltrating solid tumor tissues and draining lymph nodes of Head and Neck Squamous Cell Carcinoma (HNSCC) show an impaired immune response. In addition to an attenuated secretion of IFN-α little is known about other HNSCC-induced functional alterations in PDCs. Particular objectives in this project were to gain new insights regarding tumor-induced phenotypical and functional alterations in the PDC population. We showed by FACS analysis and RT-PCR that HNSCC orchestrates an as yet unknown subpopulation exhibiting functional autonomy in-vitro and in-vivo besides bearing phenotypical resemblance to PDCs and T cells. A subset, positive for the PDC markers CD123, BDCA-2, HLA-DR and the T cell receptor αß (TCR-αß) was significantly induced subsequent to stimulation with HNSCC in-vitro (p = 0.009) and also present in metastatic lymph nodes in-vivo. This subgroup could be functionally distinguished due to an enhanced production of IL-2 (p = 0.02), IL-6 (p = 0.0007) and TGF-ß (not significant). Furthermore, after exposure to HNSCC cells, mRNA levels revealed a D-J-beta rearrangement of the TCR-beta chain besides a strong enhancement of the CD3ε chain in the PDC population. Our data indicate an interface between the PDC and T cell lineage. These findings will improve our understanding of phenotypical and functional intricacies concerning the very heterogeneous PDC population in-vivo.

Immunomodulatory effects of Sanglifehrin A in the innate and acquired immune response of neonatal whole blood cells.

Previous investigations have shown the immunosuppressive activity of the immunophilin-binding macrolide Sanglifehrin A (SFA). In adults, SFA also exerts anti-inflammatory properties in lipopolysaccharide (LPS)-stimulated whole blood cultures. It was the aim of this study to investigate whether the unique properties of SFA are also present in the neonatal immune system, as neonates are susceptible to serious infection due to an immaturity of immune responses. We used a whole blood assay to investigate the impact of SFA on T-cell proliferation and secretion of T-cell cytokines upon Anti-CD3/Anti-CD28 costimulation. In addition, interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) secretion was assessed in whole blood monocytes after LPS stimulation. Furthermore, the influence of SFA on LPS-induced signal transduction pathways, specifically the activity of p42/44, p38 and Ap-1, was assessed in neonatal PBMCs. Neonatal cord blood lymphocytes were found to have a diminished IL-4, IL-6, TNF-alpha and interferon-gamma (IFN-gamma) production upon Anti-CD3/Anti-CD28 costimulation compared to adult whole blood lymphocytes. In contrast, no significant differences were noted for either IL-2 production or proliferation of CD4+ cells. Upon addition of 1000nM SFA to neonatal whole blood cultures, a significant inhibition of both, T-cell cytokine secretion and proliferation was demonstrated. In line with data from adult whole blood cultures, SFA proved to be a strong inhibitor of LPS-induced expression of IL-6 and TNF-alpha in the neonatal whole blood system. In signal transduction studies of the LPS pathway, a potent inhibition of the protein kinase p42/44 was demonstrated. SFA was also shown to block nuclear translocation of the transcription factor Ap-1. SFA was proved to have inhibitory effects on innate and acquired immune response of neonatal whole blood cells. The protein kinase p42/44 and the transcription factor Ap-1 were demonstrated to be potential key molecules for the anti-inflammatory effect of SFA.

Effects of ciclosporin A, tacrolimus and sirolimus on cytokine production in neonatal immune cells.

BACKGROUND: It was the aim of this study to evaluate the effects of the well-known immunosuppressive drugs ciclosporin A (CsA), tacrolimus and sirolimus on the intracytoplasmic cytokine expression of neonatal immune cells. METHODS: Immunosuppressive drugs were added to whole blood cultures of neonatal cord blood samples (n = 17) and peripheral blood samples of adults (n = 17) in vitro prior to stimulation of lymphocytes with phorbol 12-myristate 13-acetate (PMA)/ionomycin or monocytes. RESULTS: Upon exposure to ciclosporin A (500 ng/mL) or tacrolimus (25 ng/mL) the number of cytokine expressing T cells was almost completely blocked in neonatal T cells while sirolimus (10 ng/mL) only inhibited intracytoplasmatic tumour necrosis factor alpha (TNF-alpha) expression (mean% positive cells; 4.0 +/- 2.1% vs. 1.09 +/- 0.6%, p = 0.003), but mildly stimulated the intracellular expression of interleukin (IL)-2 (24.4 +/- 6.5% vs. 28.1 +/- 7.1%, p = 0.041). In cord blood lymphocytes, the inhibitory effect of ciclosporin A and tacrolimus was dose-dependent (e.g. IL-2: control, 12.3 +/- 5.33%, ciclosporin A 5 ng/mL, 10.1 +/- 5.5%; 50 ng/mL, 7.1 +/- 4.7%; 500 ng/mL, 1.2 +/- 0.3%; tacrolimus 0.25 ng/mL, 9.3 +/- 4.9%; 2.5 ng/mL, 6.1 +/- 3.3%; 25 ng/mL, 1.0 +/- 0.6%), while the function of adult lymphocytes was only impaired at high doses of both compounds. In contrast, the number of cytokine expressing monocytes was not influenced by ciclosporin A and tacrolimus except for a minor decrease of TNF-alpha producing neonatal monocytes after addition of tacrolimus (17.9% vs. 13.9%, p = 0.031). Interestingly, sirolimus was shown to inhibit intracellular IL-6 production in adults (63.1 +/- 12.7% vs. 52.0 +/- 16.0%, p = 0.005), but in neonatal monocytes intracellular IL-6 expression was stimulated (53.5 +/- 22.0% vs. 64.7 +/- 19.1%, p = 0.041). CONCLUSIONS: The potent dose-dependent inhibitory effect of ciclosporin A and tacrolimus in cord blood lymphocytes provides the basis for further studies on functional immaturity of the neonatal immune system and for future strategies to optimize umbilical cord blood transplantion. Sirolimus was demonstrated to have a distinct effect on neonatal immune cells as shown by increased expression of IL-2 in lymphocytes and IL-6 in monocytes, while only lymphocytic TNF-alpha expression was inhibited.

Immunomodulatory effect of vitamin C on intracytoplasmic cytokine production in neonatal cord blood cells.

BACKGROUND: Vitamin C (ascorbic acid) is an essential water-soluble antioxidant in cells and plasma. Besides metabolic functions, vitamin C is also known to contribute to immune homeostasis. Recently, it has been demonstrated that vitamin C has an inhibitory effect on the expression of pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-alpha) in adult whole blood cells in vitro. It has been postulated that vitamin C might be an interesting compound for modulation of an over-exuberant immune response, e.g., in patient cohorts susceptible for the development of systemic inflammatory response syndrome such as neonates. It was the aim of this study to investigate the modulatory effects of vitamin C on the production of inflammatory mediators in neonatal cord blood cells. METHODS: The intracytoplasmic production of pro-inflammatory cytokines in neonatal cord blood cells stimulated with lipopolysaccharide or phorbol 12-myristate 13-acetate/ionomycin was assessed by flow-cytometry. RESULTS: In contrast to our previous observations from adult whole blood cells, 20 mM vitamin C mildly stimulated the percentage of neonatal monocytes producing IL-6 after lipopolysaccharide stimulation (e.g., 11.3% increase compared to control, p = 0.005). In the presence of 20 mM vitamin C, even a stronger stimulatory effect was noted for the percentage of IL-8 (e.g., 46.7% increase, p < 0.001) and TNF-alpha producing neonatal monocytes (e.g., 69.2% increase, p = 0.004; n = 20). In accordance with adult data, the percentage of neonatal lymphocytes producing IL-2 after phorbol 12-myristate 13-acetate/ionomycin stimulation was dose-dependently reduced (e.g., 41.3% inhibition, p = 0.001, 20 mM vitamin C), while the percentage of TNF-alpha producing lymphocytes was mildly stimulated (e.g., 20.8% increase, p = 0.003, 20 mM vitamin C). CONCLUSIONS: Interestingly, vitamin C was demonstrated to enhance pro-inflammatory responses in CD14(+) cord blood cells while only intracellular IL-2 production in CD3(+) cells was diminished. These data suggest that vitamin C differentially influences intracytoplasmic cytokine production in adults and neonates, and further studies are needed to elucidate the underlying mechanisms of this selective immunomodulation.