Expression of the tumor antigens NY-ESO-1, tyrosinase, MAGE-A3, and TPTE in pediatric and adult melanoma: a retrospective case control study.

Tumor-associated antigens (TAAs) are potential targets for T cell-based immunotherapy approaches in cutaneous melanoma. BNT111, an investigational lipoplex-formulated mRNA-based therapeutic cancer vaccine encoding melanoma TAAs NY-ESO-1, tyrosinase, MAGE-A3, and TPTE, is undergoing clinical testing in adults. Expression of these TAAs in pediatric melanoma is unclear but is a prerequisite for feasibility of this treatment approach in children with melanoma. Our main objective was to characterize expression of those TAAs in pediatric melanomas compared to control cohorts. In this retrospective case control study, protein and transcript expression of NY-ESO-1, tyrosinase, MAGE-A3, and TPTE were analyzed in a cohort of 25 pediatric melanomas, 31 melanomas of young adults, 29 adult melanomas, and 30 benign melanocytic nevi in children using immunohistochemical staining and digital pathology (QuPath) and reverse transcription quantitative PCR. Based on IHC analysis, pediatric melanomas expressed tyrosinase (100.0%), TPTE (44.0%), MAGE-A3 (12.0%), and NY-ESO-1 (8.0%). Young adult melanomas expressed tyrosinase (96.8%), NY-ESO-1 (19.4%), MAGE-A3 (19.4%), and TPTE (3.2%). Adult melanomas expressed tyrosinase (86.2%), MAGE-A3 (75.9%), NY-ESO-1 (48.3%), and TPTE (48.3%). Childhood melanocytic nevi only expressed tyrosinase (93.3%). Expression prevalence of individual TAAs did not differ between subtypes of pediatric melanoma, and no association with prognosis was found. All four TAAs were expressed in pediatric melanoma, albeit NY-ESO-1 and MAGE-A3 to a lesser extent than in adult melanoma. These data support the possibility of investigating vaccines targeting these TAAs for the treatment of pediatric melanoma.

Functional EPAS1/HIF2A missense variant is associated with hematocrit in Andean highlanders.

Hypoxia-inducible factor pathway genes are linked to adaptation in both human and nonhuman highland species. EPAS1, a notable target of hypoxia adaptation, is associated with relatively lower hemoglobin concentration in Tibetans. We provide evidence for an association between an adaptive EPAS1 variant (rs570553380) and the same phenotype of relatively low hematocrit in Andean highlanders. This Andean-specific missense variant is present at a modest frequency in Andeans and absent in other human populations and vertebrate species except the coelacanth. CRISPR-base-edited human cells with this variant exhibit shifts in hypoxia-regulated gene expression, while metabolomic analyses reveal both genotype and phenotype associations and validation in a lowland population. Although this genocopy of relatively lower hematocrit in Andean highlanders parallels well-replicated findings in Tibetans, it likely involves distinct pathway responses based on a protein-coding versus noncoding variants, respectively. These findings illuminate how unique variants at EPAS1 contribute to the same phenotype in Tibetans and a subset of Andean highlanders despite distinct evolutionary trajectories.

cIAP1/2 Antagonism Induces Antigen-Specific T Cell-Dependent Immunity.

Checkpoint blockade immunotherapy has failed in pancreatic cancer and other poorly responsive tumor types in part due to inadequate T cell priming. Naive T cells can receive costimulation not only via CD28 but also through TNF superfamily receptors that signal via NF-κB. Antagonists of the ubiquitin ligases cellular inhibitor of apoptosis protein (cIAP)1/2, also called second mitochondria-derived activator of caspases (SMAC) mimetics, induce degradation of cIAP1/2 proteins, allowing for the accumulation of NIK and constitutive, ligand-independent activation of alternate NF-κB signaling that mimics costimulation in T cells. In tumor cells, cIAP1/2 antagonists can increase TNF production and TNF-mediated apoptosis; however, pancreatic cancer cells are resistant to cytokine-mediated apoptosis, even in the presence of cIAP1/2 antagonism. Dendritic cell activation is enhanced by cIAP1/2 antagonism in vitro, and intratumoral dendritic cells show higher expression of MHC class II in tumors from cIAP1/2 antagonism-treated mice. In this study, we use in vivo mouse models of syngeneic pancreatic cancer that generate endogenous T cell responses ranging from moderate to poor. Across multiple models, cIAP1/2 antagonism has pleiotropic beneficial effects on antitumor immunity, including direct effects on tumor-specific T cells leading to overall increased activation, increased control of tumor growth in vivo, synergy with multiple immunotherapy modalities, and immunologic memory. In contrast to checkpoint blockade, cIAP1/2 antagonism does not increase intratumoral T cell frequencies. Furthermore, we confirm our previous findings that even poorly immunogenic tumors with a paucity of T cells can experience T cell-dependent antitumor immunity, and we provide transcriptional clues into how these rare T cells coordinate downstream immune responses.

SARS-CoV-2 infects human brain organoids causing cell death and loss of synapses that can be rescued by treatment with Sofosbuvir.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which was rapidly declared a pandemic by the World Health Organization (WHO). Early clinical symptomatology focused mainly on respiratory illnesses. However, a variety of neurological manifestations in both adults and newborns are now well-documented. To experimentally determine whether SARS-CoV-2 could replicate in and affect human brain cells, we infected iPSC-derived human brain organoids. Here, we show that SARS-CoV-2 can productively replicate and promote death of neural cells, including cortical neurons. This phenotype was accompanied by loss of excitatory synapses in neurons. Notably, we found that the U.S. Food and Drug Administration (FDA)-approved antiviral Sofosbuvir was able to inhibit SARS-CoV-2 replication and rescued these neuronal alterations in infected brain organoids. Given the urgent need for readily available antivirals, these results provide a cellular basis supporting repurposed antivirals as a strategic treatment to alleviate neurocytological defects that may underlie COVID-19- related neurological symptoms.

cIAP1/2 antagonism eliminates MHC class I-negative tumors through T cell-dependent reprogramming of mononuclear phagocytes.

Loss of major histocompatibility complex (MHC) class I and interferon-γ (IFN-γ) sensing are major causes of primary and acquired resistance to checkpoint blockade immunotherapy. Thus, additional treatment options are needed for tumors that lose expression of MHC class I. The cellular inhibitor of apoptosis proteins 1 and 2 (cIAP1/2) regulate classical and alternative nuclear factor κB (NF-κB) signaling. Induction of noncanonical NF-κB signaling with cIAP1/2 antagonists mimics costimulatory signaling, augmenting antitumor immunity. We show that induction of noncanonical NF-κB signaling induces T cell-dependent immune responses, even in ß2-microglobulin (ß2M)-deficient tumors, demonstrating that direct CD8 T cell recognition of tumor cell-expressed MHC class I is not required. Instead, T cell-produced lymphotoxin reprograms both mouse and human macrophages to be tumoricidal. In wild-type mice, but not mice incapable of antigen-specific T cell responses, cIAP1/2 antagonism reduces tumor burden by increasing phagocytosis of live tumor cells. Efficacy is augmented by combination with CD47 blockade. Thus, activation of noncanonical NF-κB stimulates a T cell-macrophage axis that curtails growth of tumors that are resistant to checkpoint blockade because of loss of MHC class I or IFN-γ sensing. These findings provide a potential mechanism for controlling checkpoint blockade refractory tumors.

Neoleukin-2 enhances anti-tumour immunity downstream of peptide vaccination targeted by an anti-MHC class II VHH.

Cancer-specific mutations can lead to peptides of unique sequence presented on MHC class I to CD8 T cells. These neoantigens can be potent tumour-rejection antigens, appear to be the driving force behind responsiveness to anti-CTLA-4 and anti-PD1/L1-based therapies and have been used to develop personalized vaccines. The platform for delivering neoantigen-based vaccines has varied, and further optimization of both platform and adjuvant will be necessary to achieve scalable vaccine products that are therapeutically effective at a reasonable cost. Here, we developed a platform for testing potential CD8 T cell tumour vaccine candidates. We used a high-affinity alpaca-derived VHH against MHC class II to deliver peptides to professional antigen-presenting cells. We show in vitro and in vivo that peptides derived from the model antigen ovalbumin are better able to activate naive ovalbumin-specific CD8 T cells when conjugated to an MHC class II-specific VHH when compared with an irrelevant control VHH. We then used the VHH-peptide platform to evaluate a panel of candidate neoantigens in vivo in a mouse model of pancreatic cancer. None of the candidate neoantigens tested led to protection from tumour challenge; however, we were able to show vaccine-induced CD8 T cell responses to a melanoma self-antigen that was augmented by combination therapy with the synthetic cytokine mimetic Neo2/15.

Altered Binding of Tumor Antigenic Peptides to MHC Class I Affects CD8+ T Cell-Effector Responses.

T-cell priming occurs when a naïve T cell recognizes cognate peptide-MHC complexes on an activated antigen-presenting cell. The circumstances of this initial priming have ramifications on the fate of the newly primed T cell. Newly primed CD8+ T cells can embark onto different trajectories, with some becoming short-lived effector cells and others adopting a tissue resident or memory cell fate. To determine whether T-cell priming influences the quality of the effector T-cell response to tumors, we used transnuclear CD8+ T cells that recognize the melanoma antigen TRP1 using TRP1high or TRP1low TCRs that differ in both affinity and fine specificity. From a series of altered peptide ligands, we identified a point mutation (K8) in a nonanchor residue that, when analyzed crystallographically and biophysically, destabilized the peptide interaction with the MHC binding groove. In vitro, the K8 peptide induced robust proliferation of both TRP1high and TRP1low CD8+ T cells but did not induce expression of PD-1. Cytokine production from K8-stimulated TRP1 cells was minimal, whereas cytotoxicity was increased. Upon transfer into B16 tumor-bearing mice, the reference peptide (TRP1-M9)- and K8-stimulated TRP1 cells were equally effective at controlling tumor growth but accomplished this through different mechanisms. TRP1-M9-stimulated cells produced more IFNγ, whereas K8-stimulated cells accumulated to higher numbers and were more cytotoxic. We, therefore, conclude that TCR recognition of weakly binding peptides during priming can skew the effector function of tumor-specific CD8+ T cells.

Radiation and Local Anti-CD40 Generate an Effective in situ Vaccine in Preclinical Models of Pancreatic Cancer.

Radiation therapy induces immunogenic cell death, which can theoretically stimulate T cell priming and induction of tumor-specific memory T cell responses, serving as an in situ vaccine. In practice, this abscopal effect is rarely observed. We use two mouse models of pancreatic cancer to show that a single dose of stereotactic body radiation therapy (SBRT) synergizes with intratumoral injection of agonistic anti-CD40, resulting in regression of non-treated contralateral tumors and formation of long-term immunologic memory. Long-term survival was not observed when mice received multiple fractions of SBRT, or when TGFß blockade was combined with SBRT. SBRT and anti-CD40 was so effective at augmenting T cell priming, that memory CD8 T cell responses to both tumor and self-antigens were induced, resulting in vitiligo in long-term survivors.

Targeting Cytokine Therapy to the Pancreatic Tumor Microenvironment Using PD-L1-Specific VHHs.

Cytokine-based therapies for cancer have not achieved widespread clinical success because of inherent toxicities. Treatment for pancreatic cancer is limited by the dense stroma that surrounds tumors and by an immunosuppressive tumor microenvironment. To overcome these barriers, we developed constructs of single-domain antibodies (VHHs) against PD-L1 fused with IL-2 and IFNγ. Targeting cytokine delivery in this manner reduced pancreatic tumor burden by 50%, whereas cytokines fused to an irrelevant VHH, or blockade of PD-L1 alone, showed little effect. Targeted delivery of IL-2 increased the number of intratumoral CD8+ T cells, whereas IFNγ reduced the number of CD11b+ cells and skewed intratumoral macrophages toward the display of M1-like characteristics. Imaging of fluorescent VHH-IFNγ constructs, as well as transcriptional profiling, demonstrated targeting of IFNγ to the tumor microenvironment. Many tumors and tumor-infiltrating myeloid cells express PD-L1, rendering them potentially susceptible to this form of targeted immunotherapy. Cancer Immunol Res; 6(4); 389-401. ©2018 AACR.

IAP Antagonists Enhance Cytokine Production from Mouse and Human iNKT Cells.

Inhibitor of apoptosis protein (IAP) antagonists are in clinical trials for a variety of cancers, and mouse models show synergism between IAP antagonists and anti-PD-1 immunotherapy. Although IAP antagonists affect the intrinsic signaling of tumor cells, their most pronounced effects are on immune cells and the generation of antitumor immunity. Here, we examined the effects of IAP antagonism on T-cell development using mouse fetal thymic organ culture and observed a selective loss of iNKT cells, an effector cell type of potential importance for cancer immunotherapy. Thymic iNKT-cell development probably failed due to increased strength of TCR signal leading to negative selection, given that mature iNKT cells treated with IAP antagonists were not depleted, but had enhanced cytokine production in both mouse and human ex vivo cultures. Consistent with this, mature mouse primary iNKT cells and iNKT hybridomas increased production of effector cytokines in the presence of IAP antagonists. In vivo administration of IAP antagonists and α-GalCer resulted in increased IFNγ and IL-2 production from iNKT cells and decreased tumor burden in a mouse model of melanoma lung metastasis. Human iNKT cells also proliferated and increased IFNγ production dramatically in the presence of IAP antagonists, demonstrating the utility of these compounds in adoptive therapy of iNKT cells. Cancer Immunol Res; 6(1); 25-35. ©2017 AACR.

Cellulose Acetate Phthalate and Antiretroviral Nanoparticle Fabrications for HIV Pre-Exposure Prophylaxis.

To adequately reduce new HIV infections, development of highly effective pre-exposure prophylaxis (PrEP) against HIV infection in women is necessary. Cellulose acetate phthalate (CAP) is a pH sensitive polymer with HIV-1 entry inhibitory properties. Dolutegravir (DTG) is an integrase strand transfer inhibitor with potent antiretroviral activity. DTG delivered in combination with CAP may significantly improve current PrEP against HIV. In the present study the development of DTG-loaded CAP nanoparticles incorporated in thermosensitive (TMS) gel at vaginal pH 4.2 and seminal fluid pH 7.4 is presented as proof-of-concept for improved PrEP. Water-oil-in-water homogenization was used to fabricate DTG-loaded CAP nanoparticles (DTG-CAP-NPs). Size, polydispersity, and morphological analyses illustrate that DTG-CAP-NPs were smooth and spherical, ≤200 nm in size, and monodispersed with a polydispersity index PDI ≤ 0.2. The drug encapsulation (EE%) and release profile of DTG-CAP-NPs was determined by HPLC analysis. The EE% of DTG in DTG-CAP-NPs was evaluated to be ∼70%. The thermal sensitivity of the TMS gel was optimized and the pH dependency was evaluated by rheological analysis. DTG release studies in TMS gel revealed that DTG-CAP-NPs were stable in TMS gel at pH 4.2 while DTG-CAP-NPs in TMS gel at pH 7.4 rapidly release DTG (≥80% release within 1 h). Cytotoxicity studies using vaginal cell lines revealed that DTG-CAP-NPs were relatively non-cytotoxic at concentration <1 µg/mL. Confocal microscopic studies illustrate that ≥98% cells retained DTG-CAP-NPs intracellularly over seven days. Antiretroviral drug loaded nanocellulose fabrications in TMS gel delivered intravaginally may enhance both microbicidal and antiretroviral drug efficacy and may present a novel option for female PrEP against HIV.

Topical Tenofovir Disoproxil Fumarate Nanoparticles Prevent HIV-1 Vaginal Transmission in a Humanized Mouse Model.

Preexposure prophylaxis (PrEP) with 1% tenofovir (TFV) vaginal gel has failed in clinical trials. To improve TFV efficacy in vaginal gel, we formulated tenofovir disoproxil fumarate nanoparticles in a thermosensitive (TMS) gel (TDF-NP-TMS gel). TDF-NPs were fabricated using poly(lactic-co-glycolic acid) (PLGA) polymer and an ion-pairing agent by oil-in-water emulsification. The efficacy of TDF-NP-TMS gel was tested in humanized bone marrow-liver-thymus (hu-BLT) mice. Hu-BLT mice in the treatment group (Rx; n = 15) were administered TDF-NP-TMS gel intravaginally, having TDF at 0.1%, 0.5%, and 1% (wt/vol) concentrations, whereas the control (Ctr; n = 8) group received a blank TMS gel. All Rx mice (0.1% [n = 4], 0.5% [n = 6], and 1% [n = 5]) were vaginally challenged with two transmitted/founder (T/F) HIV-1 strains (2.5 × 10(5) 50% tissue culture infectious doses). Rx mice were challenged at 4 h (0.1%), 24 h (0.5%), and 7 days (1%) posttreatment (p.t.) and Ctr mice were challenged at 4 h p.t. Blood was drawn weekly for 4 weeks postinoculation (p.i.) for plasma viral load (pVL) using reverse transcription-quantitative PCR. Ctr mice had positive pVL within 2 weeks p.i. Rx mice challenged at 4 h and 24 h showed 100% protection and no detectable pVL throughout the 4 weeks of follow-up (P = 0.009; Mantel-Cox test). Mice challenged at 7 days were HIV-1 positive at 14 days p.i. Further, HIV-1 viral RNA (vRNA) in vaginal and spleen tissues of Rx group mice with negative pVL were examined using an in situ hybridization (ISH) technique. The detection of vRNA was negative in all Rx mice studied. The present studies elucidate TDF-NP-TMS gel as a long-acting, coitus-independent HIV-1 vaginal protection modality.

Thermosensitive Gel Containing Cellulose Acetate Phthalate-Efavirenz Combination Nanoparticles for Prevention of HIV-1 Infection.

The objective of this investigation was to develop and evaluate a nano-microbicide containing a combination of cellulose acetate phthalate (HIV-1 entry inhibitor) and efavirenz (anti-HIV agent) for HIV prophylaxis. Cellulose acetate phthalate-efavirenz combination nanoparticles (CAP-EFV-NPs) were fabricated by the nanoprecipitation method and were characterized for particle size, zeta potential and encapsulation efficiency of efavirenz. CAP-EFV-NPs were incorporated into a thermosensitive gel (CAP-EFV-NP-Gel). CAP-EFV-NPs, CAP-EFV-NP-Gel and efavirenz solution were evaluated for cytotoxicity to HeLa cells and for in vitro short-term (1-day) and long-term (3-day) prophylaxis against HIV-1 infection in TZM-bl cells. CAP-EFV-NPs had size < 100 nm, negative surface charge and encapsulation efficiency of efavirenz was > 98%. CAP-EFV-NPs and CAP-EFV-NP-Gel were significantly less toxic (P < 0.01) to HeLa cells as compared to efavirenz solution. CAP-EFV-NPs showed significantly higher prophylactic activity (P < 0.01) against HIV-1 infection to TZM-bl cells as compared to efavirenz solution and blank CAP nanoparticles. CAP-EFV-NP-Gel can be a promising nano-microbicide for long-term HIV prophylaxis.

Immunomodulatory effects of 25-hydroxyvitamin D3 on monocytic cell differentiation and influence of vitamin D3 polymorphisms in type 1 diabetes.

BACKGROUND: Preventive measures and a causal therapy for type 1 diabetes (T1D) remain elusive. An imbalance between different dendritic cells (DC) with increased immunogenic DC and decreased tolerogenic DC (tDC) may lead to T1D. Furthermore, 25(OH)D3 is associated with less adverse effects than 1,25(OH)2D3. PURPOSE: The present study was performed to clarify the remaining issues about the cellular effects of 25(OH)D3 in patients with T1D and the role of genetic polymorphisms of the vitamin D3 (VD3) metabolism on a functional cellular level. MATERIALS AND METHODS: Twelve patients with T1D were case-matched to twelve healthy controls (HC). Monocytes (MC) were either not supplemented or supplemented with 25(OH)D3 in vitro and phenotyped with fluorescence-activated cell sorting. In vitro synthesis and plasma levels of 25(OH)D3 and 1,25(OH)2D3 were analyzed as well as twelve gene polymorphisms of the VD3 metabolism. RESULTS: 25(OH)D3 significantly inhibited differentiation of MC into DC and led to an increase of intermediate cells (IC), which show a similar phenotype as tDC. The patient with a recent onset of T1D showed a higher increase in MC and IC compared to patients with long-standing T1D. There were significant differences for the increase of IC with supplementation of 25(OH)D3 between different genotypes within the polymorphisms of VDR-BsmI-rs1544410, VDR-TaqI-rs731236 and CYP24A1-rs927650. CONCLUSION: This study suggests that 25(OH)D3 shows immunomodulatory effects on a cellular level in patients with T1D and HC by inhibiting the differentiation of MC into DC and promoting the formation of IC, which are similar to tDC, thereby shifting immunity to self-tolerance. The potency of 25(OH)D3 did not differ between patients with T1D and HC. Increased plasma levels of 25(OH)D3 may inhibit a proinflammatory cell milieu. Despite of the limited patient number, this study generates the hypothesis that the immunmodulatory effects may be influenced by genotypes of the VDR and CYP24A1 illustrating their functional role in T1D susceptibility, which is worth further investigation.

Development and validation of a simple and isocratic reversed-phase HPLC method for the determination of rilpivirine from tablets, nanoparticles and HeLa cell lysates.

In the present investigation, a simple and isocratic HPLC-UV method was developed and validated for determination of rilpivirine (RPV) from dosage forms (tablets and nanoparticles) and biological matrices like HeLa cell lysates. The separation and analysis of RPV was carried out under isocratic conditions using (a) a Gemini reversed-phase C18 column (5 µm; 4.6 × 150 mm) maintained at 35°C, (b) a mobile phase consisting of a mixture of acetonitrile and 25 m m potassium dihydrogen phosphate (in the ratio 50:50 v/v) at a flow rate of 0.6 mL/min and (c) atazanavir as an internal standard. The total run time was 17 min and the analysis of RPV and internal standard was carried out at 290 nm. The method was found to be linear (r(2) value > 0.998), specific, accurate and precise over the concentration range of 0.025-2 µg/mL. The lower limit of quantification was 0.025 µg/mL, the limit of detection was 0.008 µg/mL and the recovery of RPV was >90%. The stability of the RPV analytical method was confirmed at various conditions such as room temperature (24 h), -20°C (7 days), three freeze-thaw cycles and storage in an autosampler (4°C for 48 h). The method was successfully applied for the determination of RPV from conventional dosage forms like tablets, from polymeric nanoparticles and from biological matrices like HeLa cell lysates.

Vitamin D hydroxylases CYP2R1, CYP27B1 and CYP24A1 in renal cell carcinoma.

BACKGROUND: There is increasing evidence that vitamin D metabolites influence carcinogenesis. Besides its role in mineral homoeostasis, calcitriol, the active metabolite of vitamin D (1,25(OH)2 D3 ), is known to possess antiproliferative, proapoptotic and immunomodulatory effects in cancer. Concerning the synthesis of vitamin D, the hydroxylases CYP2R1, CYP27B1 and CYP24A1 play a critical role, and the latter molecule determines the biological half-life of 1,25(OH)2 D3 , which is synthesized in the proximal renal tubules. MATERIALS AND METHODS: The adjacency of these two biological processes prompted us to investigate the gene expression of CYP2R1, CYP27B1 and CYP24A1 in patients with ccRCC. Using RT-PCR, we retrospectively compared mRNA expression profiles from human ccRCC tumour samples with those derived from the corresponding adjacent healthy tissues (n = 30). RESULTS: We observed that all three genes (CYP2R1, CYP27B1 and CYP24A1) were upregulated in tumours compared with normal tissue (P < 0·0001). Moreover, CYP24A1 displayed a significantly higher expression in tumours than CYP27B1 (P < 0·05) and CYP2R1 (P < 0·0001), whereas no differences in the expression of these genes were found in healthy renal tissue. Gene expression of CYP2R1, CYP27B1 and CYP24A did not differ between pathological classifications (TNM, grading, presence of metastasis). CONCLUSION: We thus conclude that upregulated gene expression of the catabolizing CYP24A1 as well as the synthesizing CYP2R1 and CYP27B1 may lead to a misbalance of vitamin D metabolites in ccRCC and thus contributing to its pathogenesis.

Dying of hematologic patients--treatment characteristics in a German University Hospital.

The treatment of hematologic patients in palliative situations remains a major challenge as there are special clinical needs, e.g., transfusions and the high risk for infectious complications with subsequent use of broad anti-infective treatment. Furthermore, most hematologic patients have a relatively long history of disease and are acquainted with "their" wards; that is why most are treated on these hematologic and not on specialized palliative wards. The standardized approach to the care of hematologic patients with curative treatment intention is probably not fully appropriate for palliative patients. In order to evaluate the current situation of treatment characteristics in a German university hospital, we retrospectively evaluated the medical documentation of all patients who died on a hematologic ward between 2005 and 2008. While we found a high number of chemotherapeutic, anti-infective, analgesic, and sedative treatments, of transfusions, of treatment on the intensive care units, and of invasive nature, non-somatic interventions were rather scarce. Symptom control, e.g., for bleeding events or pain, was frequently not adequately achieved. With regard to the palliative situation, a holistic approach with the maintenance of patients' autonomy and the preference for dying at home, the treatment of hematologic patients in a palliative situation has to be reconsidered.

The role of cubilin gene polymorphisms and their influence on 25(OH)D3 and 1,25(OH)2D3 plasma levels in type 1 diabetes patients.

BACKGROUND: Megalin and cubilin bind and internalize the 25(OH)D3-DBP complex in the kidney. Once the complex is internalized, 25(OH)D3 is released and activated to 1,25(OH)2D3 the ligand for the vitamin D receptor (VDR). Supporting the important role of cubilin in this process recent findings showed that cubilin deficiency results in decrease of 25(OH)D3 and 1,25(OH)2D3 plasma levels. METHODS: Two hundred T1D patients and healthy controls (n=200) were genotyped for five polymorphisms (rs3740168, rs3740165, rs1801233, rs1801229 and rs2796835) within the cubilin gene. The polymorphisms were analyzed by RFLP or real time PCR. Statistic analyses were performed by using allele-wise and genotype-wise chi2 testing by using BiAS software. A p-value<0.05 was considered as significant. RESULTS: We found that the genotype "AA" of the rs3740165 was more frequent in T1D patients compared to healthy controls (26.7% vs. 5.1%, p=4x10(-7)). Nevertheless no association between the rs3740165 polymorphism and the 25(OH)D3 or 1,25(OH)2D3 plasma levels was found. No association with the other studied polymorphisms was observed. CONCLUSION: Thus our findings reveal a novel association of the cubilin rs3740165 polymorphism with type 1 diabetes. Nevertheless how exactly this polymorphism could increase the risk to develop type 1 diabetes is subject for further investigations.

Polymorphisms of CXCR3-binding chemokines in type 1 diabetes.

Type 1 diabetes (T1D) is a T-cell-mediated autoimmune disease. Although the precise mechanisms leading to the destruction of islet beta cells are unknown, diverse studies support a role of the CXCR3-binding chemokines. A combination of a case (n = 447)-control (n = 300) and family (n = 221) analysis was performed to investigate the role of the CXCL9 (rs10336, rs3733236) and CXCL10 (rs3921, rs35795399 and rs8878) polymorphisms and their interaction with HLA high-risk haplotypes DQ2(DQA*0501-DQB*0201)-DQ8(DQA*0301-DQB*0302) in T1D. In addition, the mRNA expression of these genes and of the CXCR3 in peripheral blood mononuclear cells (PBMCs) of T1D patients was studied. In the family analysis, an overtransmission of the allele T and G of the polymorphisms rs35795399 and rs8878 in the whole group (p = 0.0520 and p = 0.0290, respectively) as well as in combination with the HLA-high risk haplotypes (p = 0.0209 and 0.0340, respectively) were observed. In addition, the haplotype rs8878G-rs35795399T was more often transmitted from parents to affected offspring, whereas the haplotype rs8878A-rs35795399C was less often transmitted (p = 0.0130 and p = 0.0201, respectively). Nevertheless these associations did not remain significant after correction for multiple testing, and they could not be corroborated in the case-control analysis. Although we did not find an association of the CXCL9 and CXCL10 polymorphisms with type 1 diabetes in the German population, we cannot discard their role in other populations or other autoimmune diseases.