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BackgroundHomozygous or compound heterozygous mutations in JAGN1 cause severe congenital neutropenia. JAGN1-mutant patients present with severe early-onset bacterial infections and most have been described as low-responders to recombinant granulocyte colony-stimulating factor (G-CSF) therapy. In a murine, hematopoietic JAGN1 knockout model, which displays susceptibility to Candida albicans infection in the absence of neutropenia, treatment with granulocyte-macrophage-CSF (GM-CSF) was able to restore the functional defect of neutrophils.PatientsWe present two unrelated patients with biallelic JAGN1 mutations, who were both treated with subcutaneous GM-CSF (sargramostim) after treatment failure to G-CSF. The first patient was an 18-year-old pregnant woman who received GM-CSF at 12 weeks of gestation up to a dose of 10 µg/kg/d for 7 days. The second patient was a 5-month-old girl who received GM-CSF for a total of 9 days at a dose of up to 20 µg/kg/d. GM-CSF did not increase neutrophil counts in our patients. Treatment was stopped when neutrophil numbers declined further, no beneficial effect was noticed, and patients presented with infections. No adverse effects were observed in either patient and the fetus. Both patients ultimately underwent successful hematopoietic stem cell transplantation.DiscussionBoth patients showed a high recurrence rate of severe infections on G-CSF treatment. GM-CSF therapy did not ameliorate the clinical phenotype, in contrast to the improvement of neutrophil function observed in the JAGN1 mouse model. No major additional extra-hematopoietic manifestations were evident in our patients.ConclusionIn two unrelated patients, GM-CSF did not have any beneficial effect on neutrophil counts. Patients with JAGN1-mutant SCN with reduced G-CSF responsiveness and elevated infection rate should be evaluated early for stem cell transplantation.

Mesenchymal stromal cells (MSC) are part of the bone marrow architecture and contribute to the homeostasis of hematopoietic stem cells. Moreover, they are known to regulate immune effector cells. These properties of MSC are pivotal under physiologic conditions, and they may aberrantly also protect malignant cells. MSCs are also found in the leukemic stem cell niche of the bone marrow and as part of the tumor microenvironment. Here, they protect malignant cells from chemotherapeutic drugs and from immune effector cells in immunotherapeutic approaches. Modulation of these mechanisms may improve the efficacy of therapeutic regimens. We investigated the effect of the histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA, Vorinostat™) on the immunomodulatory effect and cytokine profile of MSC derived from bone marrow and pediatric tumors. The immune phenotype of MSC was not markedly affected. SAHA-treated MSC showed reduced immunomodulatory effects on T cell proliferation and NK cell cytotoxicity. This effect was accompanied by an altered cytokine profile of MSC. While untreated MSC inhibited the production of certain pro-inflammatory cytokines, SAHA treatment led to a partial increase in IFNγ and TNFα secretion. These alterations of the immunosuppressive milieu might be beneficial for immunotherapeutic approaches.

Osteosarcoma is a type of bone cancer that primarily affects children and young adults. The overall 5-year survival rate for localized osteosarcoma is 70–75%, but it is only 20–30% for patients with relapsed or metastatic tumors. To investigate potential glycan-targeting structures for immunotherapy, we stained primary osteosarcomas with recombinant C-type lectin CD301 (MGL, CLEC10A) and observed moderate to strong staining on 26% of the tumors. NK92 cells expressing a CD301-CAR recognized and eliminated osteosarcoma cells in vitro. Cytotoxic activity assays correlated with degranulation and cytokine release assays. Combination with an inhibitory antibody against the immune checkpoint TIGIT (T-cell immunoreceptor with lg and ITIM domains) showed promising additional effects. Overall, this study showed, for the first time, the expression of CD301 ligands in osteosarcoma tissue and demonstrated their use as potential target structures for lectin-based immunotherapy.

BackgroundNeutrophil extracellular traps (NETs)—as double-edged swords of innate immunity—are involved in numerous processes such as infection, inflammation and tissue repair. Research on neutrophil granulocytes is limited because of their short lifetime of only a few hours. Several attempts have been made to prolong the half-life of neutrophils using cytokines and bacterial products and have shown promising results. These long-term surviving neutrophils are reported to maintain phagocytic activity and cytokine release; however, little is known regarding their capability to release NETs.MethodsWe analysed the prolongation of neutrophil survival in vitro under various culture conditions using granulocyte colony-stimulating factor (G-CSF), lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) by flow cytometry and a viability assay. Additionally, we assessed NET formation following stimulation with phorbol 12-myristate 13-acetate (PMA) by immunofluorescence staining, myeloperoxidase (MPO)-DNA sandwich-ELISA and fluorometric assays for cell-free DNA (cfDNA), neutrophil elastase (NE) and myeloperoxidase (MPO).ResultsUntreated neutrophils could form NETs after stimulation with PMA for up to 24 h. Incubation with LPS extended their ability to form NETs for up to 48 h. At 48 h, NET release of neutrophils cultured with LPS was significantly higher compared to that of untreated cells; however, no significantly different enzymatic activity of NE and MPO was observed. Similarly, incubation with G-CSF resulted in significantly higher NET release at 48 h compared to untreated cells. Furthermore, NETs showed significantly higher enzymatic activity of NE and MPO after incubation with G-CSF. Lastly, incubation with TNF-α had no influence on NET release compared to untreated cells although survival counts were altered by TNF-α.ConclusionsG-CSF, LPS or TNF-α each at low concentrations lead to prolonged survival of cultured neutrophils, resulting in considerable differences in NET formation and composition. These results provide new information for the use of neutrophils in long-term experiments for NET formation and provide novel insights for neutrophil behaviour under inflammatory conditions.

Transplantation of allogeneic hematopoietic stem cells represents an established treatment for children with high-risk leukemia. However, steroid-refractory chronic graft-versus-host disease (SR-cGvHD) represents a severe life-threatening complication, for which there is no standard therapy. After failing several lines of immunosuppressive and biological treatment, we applied an immunoablative therapy with re-transplantation of purified CD34+ donor stem cells to reset the aberrant immune system. Two pediatric patients, who had been transplanted for high-risk acute lymphoblastic leukemia, underwent the procedure. Interestingly, enough stem cells could be mobilized, harvested, and purified to be used as grafts more than one year after allogeneic transplantation under intensive immunosuppressive therapy and ongoing SR-cGvHD. With a follow-up of 8 and 22 months, respectively, both patients are without immunosuppressive therapy and do not show signs of active disease. Regeneration of skin manifestations started promptly, other damaged organs did not progress and continue to show recovery from severe fibrotic transformation. Bone marrow function is robust and T cell receptor repertoires showed polyclonal immune reconstitution. In conclusion, stem cell harvest and re-transplantation of human CD34+-selected allogeneic stem cells is possible and represents a new therapeutic option in SR-cGvHD by resetting a profoundly disturbed immune network.

Background: Mucolipidosis type II (MLII) is an ultra-rare lysosomal storage disorder caused by defective lysosomal enzyme trafficking. Clinical hallmarks are craniofacial dysmorphia, cardiorespiratory dysfunction, hepatosplenomegaly, skeletal deformities and neurocognitive retardation. Death usually occurs in the first decade of life and no cure exists. Hematopoietic stem cell transplantation (HSCT) has been performed in few MLII patients, but comprehensive follow-up data are extremely scarce. Methods: MLII diagnosis was confirmed in a female three-month-old patient with the mutations c.2213C > A and c.2220_2221dup in the GNPTAB gene. At nine months of age, the patient received HSCT from a 9/10 human leukocyte antigen (HLA)-matched unrelated donor. Results: HSCT resulted in a sustained reduction of lysosomal storage und bone metabolism markers. At six years of age, the patient showed normal cardiac function, partial respiratory insufficiency and moderate hepatomegaly, whereas skeletal manifestations had progressed. However, the patient could walk and maintained an overall good quality of life. Neurocognitive testing revealed a developmental quotient of 36%. The patient died at 6.6 years of age following a human metapneumovirus (hMPV) pneumonia. Conclusions: The exact benefit remains unclear as current literature vastly lacks comparable data on MLII natural history patients. In order to evaluate experimental therapies, in-depth prospective studies and registries of untreated MLII patients are indispensable.

Persistence of fetal cells in the circulation of the mother (fetal microchimerism, FM) is associated with increased survival and reduced relapse of children with leukemia receiving a haploidentical hematopoietic stem cell transplantation (hHSCT). NK cells play an important role in maternal tolerance towards the unborn child. In this study, 70 mother–child pairs were prospectively analyzed for the occurrence of FM, KIR genotype and HLA-C type. We found that occurrence and level of FM were influenced by three maternal genetic factors: presence of an HLA-C1 allele, absence of KIR2DL3 and presence of a cen-B/B motif. Furthermore, an HLA-C match between mother and child favored persistence of FM. NK cells from FM+ mothers showed a 40% higher specific degranulation against their filial leukemic blasts than NK cells from FM− mothers, suggesting the presence of educated maternal NK cells. Nevertheless, cytotoxicity of parental NK cells against filial leukemic blasts was independent of KIR genetics (haplotype, B content score, centromeric and telomeric KIR gene regions) and independent of FM, indicating that additional immune effector mechanisms contribute to the beneficial effect of persisting FM in hHSCT.

Hematopoietic stem cell transplantation (HSCT) represents the only curative treatment option for numerous hematologic malignancies. While the influence of donor age and the composition of the graft have already been examined in clinical and preclinical studies, little information is available on the extent to which different hematological subpopulations contribute to the dynamics of the reconstitution process and on whether and how these contributions are altered with age. In a murine model of HSCT, we therefore simultaneously tracked different cultivated and transduced hematopoietic stem and progenitor cell (HSPC) populations using a multicolor-coded barcode system (BC32). We studied a series of age-matched and age-mismatched transplantations and compared the influence of age on the reconstitution dynamics. We show that reconstitution from these cultured and assembled grafts was substantially driven by hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs) independent of age. The reconstitution patterns were polyclonal and stable in all age groups independently of the variability between individual animals, with higher output rates from MPPs than from HSCs. Our experiments suggest that the dynamics of reconstitution and the contribution of cultured and individually transduced HSPC subpopulations are largely independent of age. Our findings support ongoing efforts to expand the application of HSCT in older individuals as a promising strategy to combat hematological diseases, including gene therapy applications.

Short rotation coppice (SRC) with metal tolerant plants may attenuate the pollution of excessive elements with potential toxicity in soils, while preserving soil resources and functionality. Here, we investigated effects of 6 years phytomanagement with willow SRC on properties including heavy metal levels, toxicity tested by BioTox, microbial biomass, enzyme activities, and functional gene abundances measured by GeoChip of soils contaminated by As, Cd, Pb and Zn, as compared to the same soils under non-managed mixed grassland representing no intervention treatment (Unt). Though metal total concentrations did not differ by SRC and Unt, SRC soils had lower metal availability and toxicity, higher organic carbon, microbial biomass, phosphatase, urease and protease activities, as compared to Unt soils. Significantly reduced abundances of genes encoding resistances to various metals and antibiotics were observed in SRC, likely attributed to reduced metal selective pressure based on less heavy metal availability and soil toxicity. SRC also significantly reduced abundances of genes involved in nitrogen, phosphorus, and sulfur cycles, possibly due to the willow induced selection. Overall, while the SRC phytomanagement did not reduce the total heavy metal concentrations in soils, it decreased the heavy metal availability and soil toxicity, which in turn led to less metal selective pressure on microbial communities. The SRC phytomanagement also reduced the abundances of nutrient cycling genes from microbial communities, possibly due to intense plant nutrient uptake that depleted soil nitrogen and phosphorus availability, and thus site-specific practices should be considered to improve the soil nutrient supply for phytomanagement plants.

Introduction: The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs.Methods: Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel.Results: The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1–25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0–88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE- syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%—subcutaneous; 29%—intravenous; 1%—unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy.Conclusion: The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment.

Following bone fracture, the repair process starts with an inflammatory reaction at the fracture site. Fracture healing is disturbed when the initial inflammation is increased or prolonged, whereby, a balanced inflammatory response is anticipated to be crucial for fracture healing, because it may induce down-stream responses leading to tissue repair. However, the impact of the immune response on fracture healing remains poorly understood. Here, we investigated bone healing in NOD/scid-IL2Rγcnull mice, which exhibit severe defects in innate and adaptive immunity, by biomechanical testing, histomorphometry and micro-computed tomography. We demonstrated that NOD/scid-IL2Rγcnull mice exhibited normal skeletal anatomy and a mild bone phenotype with a slightly reduced bone mass in the trabecular compartment in comparison to immunocompetent Balb/c mice. Fracture healing was impaired in immunodeficient NOD/scid-IL2Rγcnull mice. Callus bone content was unaffected during the early healing stage, whereas it was significantly reduced during the later healing period. Concomitantly, the amount of cartilage was significantly increased, indicating delayed endochondral ossification, most likely due to the decreased osteoclast activity observed in cells isolated from NOD/scid-IL2Rγcnull mice. Our results suggest that--under aseptic, uncomplicated conditions--the immediate immune response after fracture is non-essential for the initiation of bone formation. However, an intact immune system in general is important for successful bone healing, because endochondral ossification is delayed in immunodeficient NOD/scid-IL2Rγcnull mice.

The socio-thermodynamics of a population of two competing species exhibits strong analogies with the thermodynamics of solutions and alloys of two constituents. In particular we may construct strategy diagrams akin to the phase diagrams of chemical thermodynamics, complete with regions of homogeneous mixing and miscibility gaps.

The application of autologous mesenchymal stem cells (MSC) for the treatment of bone defects requires two invasive procedures and several weeks of ex vivo cell expansion. To overcome these limitations, the administration of allogeneic MSC may be attractive, because they are anticipated to be immunoprivileged. Because preclinical studies using various animal models are conflicting with respect to the efficacy of allogeneic MSC, we investigated whether autologous and allogeneic human MSC (hMSC) are equally effective in regenerating bone in a humanized mouse model resembling the human immune system. Applying autologous and allogeneic hMSC in critically sized femoral defects, we found that allogeneic hMSC elicited a mild immune response early after implantation, whereas early angiogenic processes were similar in both treatments. At later healing time points, the transplantation of allogeneic hMSC resulted in less bone formation than autologous hMSC, associated with a reduced expression of the osteogenic factor Runx2 and impaired angiogenesis. We found by species-specific staining for collagen-type-1α2 that MSCs of either source did not synthesize new bone matrix, indicating an indirect contribution of transplanted hMSC to bone regeneration. In conclusion, our data suggest that the application of autologous hMSC is superior to that of allogeneic cells for bone defect treatment.

While the main concepts of thermodynamics are universal, the application to specific systems is not. Thus, the universal concepts combined with specific constitutive relations permit the derivation of important results in such fields as diverse as physics, chemistry, physical chemistry, chemical engineering and rheology. In all of these fields equilibrium is characterized either by a maximum of entropy or by a minimum of available free energies, depending on boundary data. In the latter case there is a compromise between the entropic tendency to grow and the energetic tendency to decrease. After some historical considerations the situation is illustrated for several specific cases: planetary atmospheres, osmosis and elastic rubber molecules, pertaining to physics, chemistry and rheology respectively. Afterwards, in the later parts of the article, thermodynamics considerations are extrapolated to remote fields, to wit evolutionary genetics and sociology.

The clinical use of bone marrow derived multipotent mesenchymal stromal cells (BM-MSCs) in different settings ranging from tissue engineering to immunotherapies has prompted investigations on the properties of these cells in a variety of other tissues. Particularly the role of MSCs in solid tumors has been the subject of many experimental approaches. While a clear phenotypical distinction of tumor associated fibroblasts (TAFs) and MSCs within the tumor microenvironment is still missing, the homing of bone marrow MSCs in tumor sites has been extensively studied. Both, tumor-promoting and tumor-inhibiting effects of BM-MSCs have been described in this context. This ambiguity requires a reappraisal of the different studies and experimental methods employed. Here, we review the current literature on tumor-promoting and tumor-inhibiting effects of BM-MSCs with a particular emphasis on their interplay with components of the immune system and also highlight a potential role of MSCs as cell of origin for certain mesenchymal tumors.

In hematopoietic stem cell transplantation for hemophagocytic lymphohistiocytosis, high transplant-related mortality after busulfan-based myeloablative regimens has been observed. Conditioning regimens with reduced toxicity based on melphalan or treosulfan are promising alternatives. We retrospectively analyzed hematopoietic stem cell transplantations in 19 hemophagocytic lymphohistiocytosis patients after conditioning with fludarabine, treosulfan, alemtuzumab, with or without thiotepa. Overall and disease-free survivals were 100% (follow up 7–31 months). Two patients required second transplant (1 after haploidentical transplantation). In 6 patients, overall donor chimerism dropped below 75% and prompted donor lymphocyte infusions. Administration of donor lymphocytes or second transplantation were significantly more frequent after transplantation from a human leukocyte antigen mismatched (9/10) versus matched (10/10) donor (P=0.018). The toxicity profile was favorable, with one veno-occlusive disease, one grade 3 graft-versus-host disease after donor lymphocyte infusion, and 2 severe viral infections (1 influenza, 1 Epstein Barr virus). In conclusion, the treosulfan-based regimen in hemophagocytic lymphohistiocytosis is effective with low toxicity and gives excellent overall and disease-free survival rates. In the future, the incidence of mixed chimerism, particularly after human leukocyte antigen mismatched donor transplants, needs to be addressed.

Detonations and flames, -just like shocks -, are steep and rapid transitions between two equilibria which represent regular singularities. The nature of the singular points is more difficult to identify than for shock waves, because of more variable-fields. After a review of the Chapman-Jouguet theory the fields of velocity, pressure, temperature, entropy and entropy production are calculated for two detonations with Mach number 7. Problems with flames in viscous gases are briefly discussed.

Systemic fungal infections are a major cause of infection-related mortality in patients with hematologic malignancies. This report addresses the case of an adolescent patient with acute lymphoblastic leukemia who underwent three allogeneic hematopoietic stem cell transplantations and developed pulmonary aspergillosis. Combination therapy with liposomal amphotericin B (L-AmB, 3 mg/kg bw/day) and caspofungin (CAS, 50 mg/day) during the first allogeneic hematopoietic stem cell transplantation (HSCT) improved the pulmonary situation. After shifting the antifungal combination therapy to oral voriconazole (2 × 200 mg/day) and CAS, a new pulmonal lesion occurred alongside the improvements in the existing pulmonary aspergillosis. An antifungal combination during a second HSCT with L-AmB (3 mg/kg bw/day) and CAS showed an improvement in the pulmonary aspergillosis. A combination therapy with CAS and L-AmB (1 mg/kg bw/day) during the third HSCT led once again to progress the pulmonary aspergillosis, after increasing the L-AMB to 3 mg/kg bw/day for recovery. The presented case provides an example of how, despite severe immunosuppression, a combination of antifungal drugs administered intravenously at therapeutic dosages may be more efficient than either intravenous monotherapy or combinations of intravenous and oral antifungals in selecting pediatric and adolescent patients with proven fungal infections.

Background Natural killer cells have been demonstrated to exert remarkable graft-versus-leukemia effects after haploidentical transplantation. Acquisition of both, inhibiting and activating, receptors on developing natural killer cells is an important step in their functional maturation. Here, we report on the reconstitution of natural killer receptors after haploidentical transplantation of T-and B-cell (CD3/CD19) depleted grafts with co-transfusion of natural killer cells in children and its influence on natural killer cell activity and clinical outcome.Design and Methods We analyzed reconstitution patterns of natural killer receptors at different time intervals after haploidentical transplantation by multi-color flow cytometry. Natural killer cell activity and antibody-dependent cellular cytotoxicity was tested against cell lines and leukemic blasts in vitro. Survival was analyzed using Kaplan-Meier estimates.Results Recovery of CD56+/CD16+ cells was fast with high cytolytic activity against K562 and strong antibody-dependent cellular cytotoxicity activity against neuroblastoma and leukemic blasts as early as day 14 posttransplant. KIR reconstitution showed a predominance of KIR negative natural killer cells early after transplantation and an early reconstitution of CD158b compared to CD158a and CD158e. These differences were independent of presence or absence of the corresponding KIR ligands in donors or recipients. This reconstitution pattern was associated with a higher relapse probability of patients homozygous for HLA-C1-alleles compared to patients homozygous or even heterozygous for HLA-C2-alleles.Conclusions Our results indicate a fast recovery of functional and alloreactive natural killer cells with a constant KIR pattern after haploidentical transplantation with T- and B-cell depleted grafts. Moreover, these natural killer cells can mediate antibody-dependent cellular cytotoxicity and therefore may allow for an early use of antibodies against residual malignant cells.

Background Conventionally, mesenchymal stem cells are functionally isolated from primary tissue based on their capacity to adhere to a plastic surface. This isolation procedure is hampered by the unpredictable influence of co-cultured hematopoietic and/or other unrelated cells and/or by the elimination of a late adhering mesenchymal stem cells subset during removal of undesired cells. To circumvent these limitations, several antibodies have been developed to facilitate the prospective isolation of mesenchymal stem cells. Recently, we described a panel of monoclonal antibodies with superior selectivity for mesenchymal stem cells, including the monoclonal antibodies W8B2 against human mesenchymal stem cell antigen-1 (MSCA-1) and 39D5 against a CD56 epitope, which is not expressed on natural killer cells.Design and Methods Bone marrow derived mesenchymal stem cells from healthy donors were analyzed and isolated by flow cytometry using a large panel of antibodies against surface antigens including CD271, MSCA-1, and CD56. The growth of mesenchymal stem cells was monitored by colony formation unit fibroblast (CFU-F) assays. The differentiation of mesenchymal stem cells into defined lineages was induced by culture in appropriate media and verified by immunostaining.Results Multicolor cell sorting and CFU-F assays showed that mesenchymal stem cells were ~90-fold enriched in the MSCA-1+CD56− fraction and ~180-fold in the MSCA-1+CD56+ fraction. Phenotype analysis revealed that the expression of CD10, CD26, CD106, and CD146 was restricted to the MSCA-1+CD56− mesenchymal stem cells subset and CD166 to MSCA-1+CD56± mesenchymal stem cells. Further differentiation of these subsets showed that chondrocytes and pancreatic-like islets were predominantly derived from MSCA-1+CD56± cells whereas adipocytes emerged exclusively from MSCA-1+CD56− cells. The culture of single sorted MSCA-1+CD56+ cells resulted in the appearance of phenotypically heterogeneous clones with distinct proliferation and differentiation capacities.Conclusions Novel mesenchymal stem cells subsets with distinct phenotypic and functional properties were identified. Our data suggest that the MSCA-1+CD56+ subset is an attractive starting population for autologous chondrocyte transplantation.

This paper presents a thermodynamic theory of light and sound. It demonstrates that extended thermodynamics permits the explicit calculation of the main part of the equations of balance of energy for photons and phonons. Wave speeds are calculated and the limiting cases of near-equilibrium and free streaming are discussed.