Decreased eukaryotic initiation factors expression upon temozolomide treatment-potential novel implications for eIFs in glioma therapy.

PURPOSE: Since glioma therapy is currently still limited until today, new treatment options for this heterogeneous group of tumours are of great interest. Eukaryotic initiation factors (eIFs) are altered in various cancer entities, including gliomas. The purpose of our study was to evaluate the potential of eIFs as novel targets in glioma treatment. METHODS: We evaluated eIF protein expression and regulation in 22 glioblastoma patient-derived xenografts (GBM PDX) after treatment with established cytostatics and with regards to mutation profile analyses of GBM PDX. RESULTS: We observed decreased expression of several eIFs upon temozolomide (TMZ) treatment independent from the phosphatidylinositol 3-kinase (PI3K)/ AKT/ mammalian target of the rapamycin (mTOR) signalling pathway. These effects of TMZ treatment were not present in TMZ-resistant PDX. Combination therapy of regorafenib and TMZ re- established the eIF/AKT/mTOR axis. CONCLUSION: Our study provides novel insights into chemotherapeutic effects on eIF regulation in gliomas and suggests that eIFs are interesting candidates for future research to improve glioma therapy.

A Profound Basic Characterization of eIFs in Gliomas: Identifying eIF3I and 4H as Potential Novel Target Candidates in Glioma Therapy.

Glioblastoma (GBM) is an utterly devastating cerebral neoplasm and current therapies only marginally improve patients' overall survival (OS). The PI3K/AKT/mTOR pathway participates in gliomagenesis through regulation of cell growth and proliferation. Since it is an upstream regulator of the rate-limiting translation initiation step of protein synthesis, controlled by eukaryotic initiation factors (eIFs), we aimed for a profound basic characterization of 17 eIFs to identify potential novel therapeutic targets for gliomas. Therefore, we retrospectively analyzed expressions of mTOR-related proteins and eIFs in human astrocytoma samples (WHO grades I-IV) and compared them to non-neoplastic cortical control brain tissue (CCBT) using immunoblot analyses and immunohistochemistry. We examined mRNA expression using qRT-PCR and additionally performed in silico analyses to observe the influence of eIFs on patients' survival. Protein and mRNA expressions of eIF3B, eIF3I, eIF4A1, eIF4H, eIF5 and eIF6 were significantly increased in high grade gliomas compared to CCBT and partially in low grade gliomas. However, short OS was only associated with high eIF3I gene expression in low grade gliomas, but not in GBM. In GBM, high eIF4H gene expression significantly correlated with shorter patient survival. In conclusion, we identified eIF3I and eIF4H as the most promising targets for future therapy for glioma patients.

Eukaryotic Translation Initiation Factor 4AI: A Potential Novel Target in Neuroblastoma.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor. Children suffering from high-risk and/or metastatic NB often show no response to therapy, and new therapeutic approaches are urgently needed. Malignant tumor development has been shown to be driven by the dysregulation of eukaryotic initiation factors (eIFs) at the translation initiation. Especially the activity of the heterotrimeric eIF4F complex is often altered in malignant cells, since it is the direct connection to key oncogenic signaling pathways such as the PI3K/AKT/mTOR-pathway. A large body of literature exists that demonstrates targeting the translational machinery as a promising anti-neoplastic approach. The objective of this study was to determine whether eIF4F complex members are aberrantly expressed in NB and whether targeting parts of the complex may be a therapeutic strategy against NB. We show that eIF4AI is overexpressed in NB patient tissue using immunohistochemistry, immunoblotting, and RT-qPCR. NB cell lines exhibit decreased viability, increased apoptosis rates as well as changes in cell cycle distribution when treated with the synthetic rocaglate CR-1-31-B, which clamps eIF4A and eIF4F onto mRNA, resulting in a translational block. Additionally, this study reveals that CR-1-31-B is effective against NB cell lines at low nanomolar doses (≤20 nM), which have been shown to not affect non-malignant cells in previous studies. Thus, our study provides information of the expression status on eIF4AI in NB and offers initial promising insight into targeting translation initiation as an anti-tumorigenic approach for NB.

Lymphopenia in primary Sjögren's syndrome is associated with premature aging of naïve CD4+ T cells.

OBJECTIVE: To investigate peripheral lymphopenia, a frequent finding in primary Sjögren's syndrome (pSS) associated with higher disease activity and increased mortality. METHODS: Prospective, cross-sectional study of consecutive patients with pSS (n = 66) and healthy controls (n = 181). Lymphocyte subsets were analysed by flow cytometry, naïve (CD45RA+) and memory (CD45RO+) CD4+ T cells were purified by MACS technology. In vitro proliferation and senescence-associated ß-galactosidase (SABG) were assessed by flow cytometry. Telomere length and TCR excision circles (TREC) were measured by real-time PCR. Telomerase activity was analysed according to the telomeric repeat amplification protocols (TRAP). RESULTS: In pSS, lymphopenia mainly affected naïve CD4+ T cells. We noted a lower frequency of proliferating naïve CD4+ T cells ex vivo and decreased homeostatic proliferation in response to IL-7 stimulation in vitro. Furthermore, naïve CD4+ T cells exhibited signs of immune cell aging including shortened telomeres, a reduction in IL-7R expression and accumulation of SABG. The senescent phenotype could be explained by telomerase insufficiency and drastically reduced levels of T-cell receptor excision circles (TRECs), indicating a history of extensive post-thymic cell division. TRECs correlated with the number of naïve CD4+ T cells linking the extend of earlier proliferation to the inability to sustain normal cell numbers. CONCLUSION: In pSS, evidence for increased proliferation of naïve CD4+ T cells earlier in life is associated with a senescent phenotype unable to sustain homeostasis. The lack of naïve CD4+ T cells forms the basis of lymphopenia frequently observed in pSS.

No Overt Clinical Immunodeficiency Despite Immune Biological Abnormalities in Patients With Constitutional Mismatch Repair Deficiency.

Immunoglobulin class-switch recombination (CSR) and somatic hypermutations (SHMs) are prerequisites for antibody and immunoglobulin receptor maturation and adaptive immune diversity. The mismatch repair (MMR) machinery, consisting of homologs of MutSα, MutLα, and MutSß (MSH2/MSH6, MLH1/PMS2, and MSH2/MSH3, respectively) and other proteins, is involved in CSR, primarily acting as a backup for nonhomologous end-joining repair of activation-induced cytidine deaminase-induced DNA mismatches and, furthermore, in addition to error-prone polymerases, in the repair of SHM-induced DNA breaks. A varying degree of antibody formation defect, from IgA or selective IgG subclass deficiency to common variable immunodeficiency and hyper-IgM syndrome, has been detected in a small number of patients with constitutional mismatch repair deficiency (CMMRD) due to biallelic loss-of-function mutations in one of the MMR genes (PMS2, MSH6, MLH1, or MSH2). To elucidate the clinical relevance of a presumed primary immunodeficiency (PID) in CMMRD, we systematically collected clinical history and laboratory data of a cohort of 15 consecutive, unrelated patients (10 not previously reported) with homozygous/compound heterozygous mutations in PMS2 (n = 8), MSH6 (n = 5), and MLH1 (n = 2), most of whom manifested with typical malignancies during childhood. Detailed descriptions of their genotypes, phenotypes, and family histories are provided. Importantly, none of the patients showed any clinical warning signs of PID (infections, immune dysregulation, inflammation, failure to thrive, etc.). Furthermore, we could not detect uniform or specific patterns of laboratory abnormalities. The concentration of IgM was increased in 3 out of 12, reduced in 3 out of 12, and normal in 6 out of 12 patients, while concentrations of IgG and IgG subclasses, except IgG4, and of IgA, and specific antibody formation were normal in most. Class-switched B memory cells were reduced in 5 out of 12 patients, and in 9 out of 12 also the CD38hiIgM- plasmablasts were reduced. Furthermore, results of next generation sequencing-based analyses of antigen-selected B-cell receptor rearrangements showed a significantly reduced frequency of SHM and an increased number of rearranged immunoglobulin heavy chain (IGH) transcripts that use IGHG3, IGHG1, and IGHA1 subclasses. T cell subsets and receptor repertoires were unaffected. Together, neither clinical nor routine immunological laboratory parameters were consistently suggestive of PID in these CMMRD patients, but previously shown abnormalities in SHM and rearranged heavy chain transcripts were confirmed.

HHV-6 Specific T-Cell Immunity in Healthy Children and Adolescents.

Objective: Primary infection with human herpes virus 6 (mainly HHV-6B) commonly occurs in the first 2 years of life leading to persistence and the possibility of virus reactivation later in life. Consequently, a specific cellular immune response is essential for effective control of virus reactivation. We have studied cell-mediated immune response to HHV-6 (U54) in healthy children and adolescents. Materials and Methods: By flow cytometry, the amount of cytokine (interferon gamma-IFN- γ, interleukin 2-IL-2, tumor necrosis factor alpha-TNF-α) secreting T-cells were measured after 10 days of pre-sensitization and 6 h of re-stimulation with mixtures of pooled overlapping peptides from U54, staphylococcal enterotoxin B (SEB, positive control), or Actin (negative control) in healthy children and adolescents without any underlying immune disorder or infectious disease. Results: All individuals showed a virus-specific response for at least one cytokine in either CD4+ or CD8+ cells. Percentages of individuals with HHV-6-specific TNF-α response in CD4+ (48% of individuals) as well as CD8+ (56% of individuals) were always the highest. Our data show significantly higher frequencies of HHV-6-specific TNF-α producing CD8+ T-cells in individuals older than 10 years of life (p = 0.033). Additionally, the frequency of HHV-6 specific TNF-α producing CD8+ T-cells positively correlated with the age of the individuals. Linear regression analysis showed a positive relation between age and frequency of HHV-6-specific TNF-α producing CD8+ T-cells. Conclusion: Results indicate that T-cell immune response against HHV-6 is commonly detectable in healthy children and adolescents with higher frequencies of antigen-specific T-cells in older children and adolescents possibly reflecting repeated stimulation by viral persistence and subclinical reactivation.

Novel Senescent Regulatory T-Cell Subset with Impaired Suppressive Function in Rheumatoid Arthritis.

OBJECTIVE: Premature senescence of lymphocytes is a hallmark of inflammatory rheumatic diseases such as rheumatoid arthritis (RA). Early T-cell aging affects conventional T-cells but is presumably not limited to this cell population; rather it might also occur in the regulatory T-cells (Tregs) compartment. In RA, Tregs fail to halt aberrant immune reactions and disease progression. Whether this is associated with early Treg senescence leading to phenotypic and functional changes of this subset is elusive so far. METHODS: Eighty-four RA patients and 75 healthy controls were prospectively enrolled into the study. Flow cytometry, magnetic-associated cell sorting, and cell culture experiments were performed for phenotypic and functional analyses of Treg subsets. T-cell receptor excision circle (TREC) levels and telomere lengths were determined using RT-PCR. RESULTS: In this paper, we describe the novel CD4+FoxP3+CD28- T-cell subset (CD28- Treg-like cells) in RA patients revealing features of both Tregs and senescent T-cells: Treg surface/intracellular markers such as CD25, CTLA-4, and PD-1 as well as FOXP3 were all expressed by CD28- Treg-like cells, and they yielded signs of premature senescence including reduced TREC levels and an accumulation of γH2AX. CD28- Treg-like could be generated in vitro by stimulation of (CD28+) Tregs with TNF-α. CD28- Treg-like cells insufficiently suppressed the proliferation of effector T-cells and yielded a pro-inflammatory cytokine profile. CONCLUSION: In conclusion, we describe a novel T-cell subset with features of Tregs and senescent non-Tregs. These cells may be linked to an aberrant balance between regulatory and effector functions in RA.

Premature senescence of T-cell subsets in axial spondyloarthritis.

OBJECTIVE: To investigate the possible occurrence of early thymic failure and premature senescence of naïve and memory T-cells in patients with axial spondyloarthritis (aSpA). METHODS: Prospective, cross-sectional study of consecutive patients with aSpA (n=51), rheumatoid arthritis (RA, n=51) and healthy controls (HCs, n=50). Demographic, clinical and laboratory parameters were collected in all patients and we isolated naïve (CD45RA(+)) and memory (CD45RO(+)) CD4(+) and CD8(+) T-cell subsets by MACS technology. T-cell receptor rearrangement excision circle (TREC) and telomere length were measured by real-time PCR. We used TRECs as a surrogate for thymus function and telomere length as an indicator of cellular senescence. Telomerase activity was analysed with the Telomeric Repeat Amplification Protocols. RESULTS: We observed a premature decline of thymic output in patients with aSpA and patients with RA compared with HCs as indicated by a reduction of TREC levels in naive T-cells (aSpA: age adjusted regression coefficient (regcoeff) for CD4(+)CD45RA(+) T-cells -2.566, p=0.023; RA regcoeff=-2.844, p=0.008). Telomere length of all CD4(+) and CD8(+) T-cell subsets was reduced in young patients with aSpA compared with HCs, whereas data for patients with RA were comparable with HCs. Telomerase activity was inversely correlated with telomere length in HCs (correlation coefficient (corcoeff)=-0.532, p<0.001) but not in patients with aSpA (corcoeff=-0.056, p=0.697) and RA (corcoeff=-0.003, p=0.982). CONCLUSIONS: Our data indicate an age-inappropriate shrinkage of thymic output, an inappropriate shortening of telomeres in young patients with aSpA and an impaired telomerase enzyme in patients with aSpA and RA.

Unrelated CD3/CD19-depleted peripheral stem cell transplantation for Hurler syndrome.

For patients with mucopolysaccharidosis type IH (MPS1-H; Hurler syndrome), early allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice. One boy and one girl aged 20.5 and 22 months, respectively, with MPS1-H received a conditioning regimen consisting of thiotepa, fludarabine, treosulfan, and ATG. Grafts were peripheral blood stem cells from unrelated donors (10/12 and 11/11 matched), that were manipulated by CD3/CD19 depletion and contained 20.3 and 28.2 × 10(6) CD34+ cells/kg body weight, respectively. Both patients achieved stable hematopoietic engraftment and stable donor chimerism. Neither acute or chronic graft-versus-host disease (GVHD) nor other severe transplant-related complications occurred. At a follow-up of 48 and 37 months, both patients are alive and well with normal levels of α-L-iduronidase and have made major neurodevelopmental progress. Treosulfan-based conditioning offers the advantage of reduced toxicity; the use of unrelated CD3/CD19-depleted peripheral stem cell grafts allows transfusion of high CD34+ cell numbers together with a "tailored" number of CD3+ cells as well as engraftment facilitating cells in order to achieve rapid hematopoietic engraftment while reducing the risk of graft rejection and GVHD. This regimen might be an additional option when unrelated donor HSCT is considered for a patient with MPS1-H.

Severe epstein-barr virus encephalitis with hemophagocytic syndrome: rapid clearance of virus following allogeneic hematopoietic stem cell transplantation from a seropositive donor.

Epstein-Barr virus-associated hemophagocytic lymphohistiocytosis with encephalitis is a life-threatening condition that requires treatment with immunochemotherapy; refractory patients are eligible for allogeneic hematopoietic stem cell transplantation. We report on an adolescent female who failed to respond to induction immunochemotherapy and was salvaged by allogeneic hematopoietic stem cell transplantation from her human leukocyte antigen-identical, Epstein-Barr virus-seropositive brother leading to rapid clearance of Epstein-Barr virus from blood and cerebrospinal fluid.