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1.
Eur Heart J Case Rep ; 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31365055

RESUMO

BACKGROUND : The aetiology of dilated cardiomyopathy (DCM) is highly heterogeneous including genetic and/or acquired (infective, toxic, immune, endocrine, and nutritional) factors. The major part of acquired DCM in developed countries is caused by either viral or autoimmune myocarditis. It is believed that the activation of the T-lymphocyte cell system is the major pathomechanism underlying autoimmune myocarditis and inflammatory DCM (DCMi). However, in the hearts of a subset of patients, a significant number of CD20+ B-lymphocytes can be detected too. Limited information exists on the role of B-cell-dependent mechanisms in the progression of DCMi. Particularly CD20+ B-lymphocytes, which can be targeted by anti-CD20+ B-lymphocytes antibodies or inhibitors, might contribute to the pathogenesis of myocardial damage beyond antibody production. CASE SUMMARY : Here, we present a case series of six patients with subacute and chronic endomyocardial biopsy-proven CD20+ B-lymphocyte-associated DCMi, where symptomatic heart failure therapy, with or without combined immunosuppressive therapy with steroid-based treatment regime, was insufficient to improve cardiac function. Five patients improved clinically several weeks after a standard infusion protocol with rituximab, a chimeric monoclonal antibody against the pan-B-cell surface molecule CD20. DISCUSSION : Our case series shows that CD20+ B-lymphocyte persistence can play a pathophysiologic role in a subset of DCMi patients and highlights the potential of targeting CD20+ B cells in patients with prominent CD20+ B-lymphocyte persistence.

2.
Sci Rep ; 9(1): 10507, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324868

RESUMO

Based on data of the SPRINT trial, American hypertension guidelines recently reduced the blood pressure goal from 140/90 mmHg to 130/80 mmHg for subjects with chronic kidney disease (CKD), whereas European guidelines recommend a systolic blood pressure (SBP) of 130-139 mmHg. The present analysis investigates whether a SBP < 130 mmHg is associated with an additional benefit in renal transplant recipients. We performed a retrospective analysis of 815 renal transplant recipients who were stratified according to mean office SBP values < 130 mmHg, 130-139 mmHg or ≥140 mmHg. Patient and graft survival was defined as composite endpoint, follow-up was limited to 120 months. Mean SBP of the follow-up was significantly associated with the composite endpoint (n = 218) with better survival for a SBP < 130 mmHg and 130-139 mmHg compared to ≥140 mmHg (p < 0.001). The differences in the combined endpoint remained significant in Cox regression analysis adjusted for age, gender and eGFR (p = 0.007, HR = 0.58, 95%CI = 0.41-0.53), but not for graft survival alone. Renal transplant recipients with SBP < 130 mmHg had a lower mortality than those with the conservative blood pressure goal <140 mmHg. These data suggest that the new AHA BP targets are safe for renal transplant recipients and - with all limitations of a retrospective analysis - might even be associated with improved outcome.

3.
Transplantation ; 2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31205263

RESUMO

BACKGROUND: Graft-versus-host disease (GvHD) presents a major cause for morbidity and mortality after allogeneic hematopoietic stem cell transplantation (alloHSCT). Rabbit anti-thymocyte globulin (rATG) treatment reduces the incidence of GvHD after alloHSCT. However, delayed immune reconstitution following rATG treatment, partly due to hampered thymic function, is being discussed. The present study aimed at elucidating possible cytotoxic effects of two commonly used rATG preparations on cultured human thymic stroma, especially thymic epithelial cells (TECs). METHODS: A primary thymic epithelial cell culture was established and the binding and cytotoxicity of two rATG preparations to the aforementioned cells were assessed by flow cytometry and immunofluorescence analyses. The release of several cytokines by cultured TSCs in response to rATG was analyzed via multiplex ELISA. RESULTS: Both preparations showed a comparable dose-dependent binding to TECs and exerted a similar complement-independent, dose-dependent cytotoxicity. rATG exposure further resulted in hampered secretion of IL-7, IL-15 and IL-6, cytokines being involved in thymic T cell development and proliferation. Pretreatment with keratinocyte growth factor (KGF) diminished rATG-induced cytotoxicity of TECs and restored their IL-7 and IL-15 secretion. CONCLUSIONS: Cytotoxic effects on TECs link a rATG-induced thymic damage to the delayed T cell reconstitution witnessed after rATG treatment. Our data support a combination treatment of rATG and thymus-protective strategies such as KGF in order to simultaneously offer sufficient GvHD prophylaxis and overcome delayed T cell reconstitution due to thymic damage.

4.
Clin J Am Soc Nephrol ; 14(7): 1056-1066, 2019 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-31213508

RESUMO

BACKGROUND AND OBJECTIVES: The prognostic value of preformed donor-specific HLA antibodies (DSA), which are only detectable by sensitive methods, remains controversial for kidney transplantation. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: The outcome of 4233 consecutive kidney transplants performed between 2012 and 2015 in 18 German transplant centers was evaluated. Most centers used a stepwise pretransplant antibody screening with bead array tests and differentiation of positive samples by single antigen assays. Using these screening results, DSA against HLA-A, -B, -C, -DRB1 and -DQB1 were determined. Data on clinical outcome and possible covariates were collected retrospectively. RESULTS: Pretransplant DSA were associated with lower overall graft survival, with a hazard ratio of 2.53 for living donation (95% confidence interval [95% CI], 1.49 to 4.29; P<0.001) and 1.59 for deceased donation (95% CI, 1.21 to 2.11; P=0.001). ABO-incompatible transplantation was associated with worse graft survival (hazard ratio, 2.09; 95% CI, 1.33 to 3.27; P=0.001) independent from DSA. There was no difference between DSA against class 1, class 2, or both. Stratification into DSA <3000 medium fluorescence intensity (MFI) and DSA ≥3000 MFI resulted in overlapping survival curves. Therefore, separate analyses were performed for 3-month and long-term graft survival. Although DSA <3000 MFI tended to be associated with both lower 3-month and long-term transplant survival in deceased donation, DSA ≥3000 MFI were only associated with worse long-term transplant survival in deceased donation. In living donation, only strong DSA were associated with reduced graft survival in the first 3 months, but both weak and strong DSA were associated with reduced long-term graft survival. A higher incidence of antibody-mediated rejection within 6 months was only associated with DSA ≥3000 MFI. CONCLUSIONS: Preformed DSA were associated with an increased risk for graft loss in kidney transplantation, which was greater in living than in deceased donation. Even weak DSA <3000 MFI were associated with worse graft survival. This association was stronger in living than deceased donation.

5.
Front Immunol ; 10: 1148, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31191530

RESUMO

Viral infections have a major impact on morbidity and mortality of immunosuppressed solid organ transplant (SOT) patients because of missing or failure of adequate pharmacologic antiviral treatment. Adoptive antiviral T-cell therapy (AVTT), regenerating disturbed endogenous T-cell immunity, emerged as an attractive alternative approach to combat severe viral complications in immunocompromised patients. AVTT is successful in patients after hematopoietic stem cell transplantation where T-cell products (TCPs) are manufactured from healthy donors. In contrast, in the SOT setting TCPs are derived from/applied back to immunosuppressed patients. We and others demonstrated feasibility of TCP generation from SOT patients and first clinical proof-of-concept trials revealing promising data. However, the initial efficacy is frequently lost long-term, because of limited survival of transferred short-lived T-cells indicating a need for next-generation TCPs. Our recent data suggest that Rapamycin treatment during TCP manufacture, conferring partial inhibition of mTOR, might improve its composition. The aim of this study was to confirm these promising observations in a setting closer to clinical challenges and to deeply characterize the next-generation TCPs. Using cytomegalovirus (CMV) as model, our next-generation Rapamycin-treated (Rapa-)TCP showed consistently increased proportions of CD4+ T-cells as well as CD4+ and CD8+ central-memory T-cells (TCM). In addition, Rapamycin sustained T-cell function despite withdrawal of Rapamycin, showed superior T-cell viability and resistance to apoptosis, stable metabolism upon activation, preferential expansion of TCM, partial conversion of other memory T-cell subsets to TCM and increased clonal diversity. On transcriptome level, we observed a gene expression profile denoting long-lived early memory T-cells with potent effector functions. Furthermore, we successfully applied the novel protocol for the generation of Rapa-TCPs to 19/19 SOT patients in a comparative study, irrespective of their history of CMV reactivation. Moreover, comparison of paired TCPs generated before/after transplantation did not reveal inferiority of the latter despite exposition to maintenance immunosuppression post-SOT. Our data imply that the Rapa-TCPs, exhibiting longevity and sustained T-cell memory, are a reasonable treatment option for SOT patients. Based on our success to manufacture Rapa-TCPs from SOT patients under maintenance immunosuppression, now, we seek ultimate clinical proof of efficacy in a clinical study.

6.
Biomaterials ; 216: 119283, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31247481

RESUMO

Extracellular matrix (ECM) provides a scaffold for cells and tissues, but also supports organogenesis and tissue remodeling. The required instructive properties of the ECM to interact with cells depend on matrix architecture, structural proteins and functional matrix components such as growth factors, providing spatial, chemical and functional cues. Decellularized ECM (dECM) has been proposed as an instructive material that promotes tissue regeneration. We investigated the instructive ECM elements preserved in dECM and necessary to promote endothelial differentiation of human induced pluripotent stem cells (hiPSC). We show that detergent-decellularized human kidney ECM remains structurally intact and carries a number of heparin-binding growth factors, including FGF2, VEGF, BMP2, HGF, EGF, PDGF-BB and TGFß, albeit at reduced levels compared to native tissues. Clearance of these heparin-binding factors, or heparan-sulfate proteoclycans from ECM resulted in massively reduced differentiation of hiPSC, suggesting that remaining structural dECM proteins such as laminin, collagen or fibronectin alone are not instructive. In contrast, replenishing dECM with VEGF replaced medium-supplemented VEGF and resulted in more efficient differentiation of hiPSC into endothelial cells, and even in the absence of other culture-supplemented differentiation factors dECM alone was superior to geltrex. In conclusion, conditioning of dECM with specific growth factors acting as functional cues may allow to generate functional niches by selective promotion of cell attachment, survival and differentiation.

7.
Transpl Int ; 2019 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-31099091

RESUMO

Expansions of donor pools have a controversial impact on healthcare expenditures. The aim of this study was to investigate the emerging costs of expanded criteria donor (ECD) kidney transplantations (KT) and to identify independent risk factors for increased transplant-related costs. We present a retrospective explorative analysis of hospital costs and reimbursements of KTs performed between 2012 and 2016 in a German university hospital. A total of 174 KTs were examined, including 92 (52.9%) ECD organ transplantations. The ECD group comprised 43 (24.7%) 'old-for-old' transplantations. Median healthcare costs were 19 570€ (IQR 18 735-27 405€) in the standard criteria donor (SCD) group versus 25 478€ (IQR 19 957-29 634€) in the ECD group (+30%; P = 0.076). 'Old-for-old' transplantations showed the highest healthcare expenditures [26 702€ (19 570-33 940€)]. Irrespective of the allocation group, transplant-related costs increased significantly in obese (+6221€; P = 0.009) and elderly recipients (+6717€; P = 0.019), in warm ischaemia time exceeding 30 min (+3212€; P = 0.009) and in kidneys with DGF or surgical complications (+8976€ and +10 624€; both P < 0.001). Transplantation of ECD organs is associated with incremental costs, especially in elderly and obese recipients. A critical patient selection, treatment of obesity before KT and keeping warm ischaemia times short seem to be crucial, in order to achieve a cost-effective KT regardless of the allocation group.

8.
Front Immunol ; 10: 593, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31019503

RESUMO

Influenza vaccination is a common approach to prevent seasonal and pandemic influenza. Pre-existing antibodies against close viral strains might impair antibody formation against previously unseen strains-a process called original antigenic sin. The role of this pre-existing cellular immunity in this process is, despite some hints from animal models, not clear. Here, we analyzed cellular and humoral immunity in healthy individuals before and after vaccination with seasonal influenza vaccine. Based on influenza-specific hemagglutination inhibiting (HI) titers, vaccinees were grouped into HI-negative and -positive cohorts followed by in-depth cytometric and TCR repertoire analysis. Both serological groups revealed cross-reactive T-cell memory to the vaccine strains at baseline that gave rise to the majority of vaccine-specific T-cells post vaccination. On the contrary, very limited number of vaccine-specific T-cell clones was recruited from the naive pool. Furthermore, baseline quantity of vaccine-specific central memory helper T-cells and clonotype richness of this population directly correlated with the vaccination efficacy. Our findings suggest that the deliberate recruitment of pre-existing cross-reactive cellular memory might help to improve vaccination outcome.

9.
Front Immunol ; 10: 767, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31024575

RESUMO

Reactivation of the BK polyomavirus is known to lead to severe complications in kidney transplant patients. The current treatment strategy relies on decreasing the immunosuppression to allow the immune system to clear the virus. Recently, we demonstrated a clear association between the resolution of BKV reactivation and reconstitution of BKV-specific CD4+ T-cells. However, which factors determine the duration of viral infection clearance remains so far unclear. Here we apply a combination of in-depth multi-parametric flow cytometry and NGS-based CDR3 beta chain receptor repertoire analysis of BKV-specific T-cells to a cohort of 7 kidney transplant patients during the clinical course of BKV reactivation. This way we followed TCR repertoires at single clone levels and functional activity of BKV-specific T-cells during the resolution of BKV infection. The duration of BKV clearance did not depend on the number of peripheral blood BKV-specific T-cells nor on a few immunodominant BKV-specific T-cell clones. Rather, the T-cell receptor repertoire diversity and exhaustion status of BKV-specific T-cells affected the duration of viral clearance: high clonotype diversity and lack of PD1 and TIM3 exhaustion markers on BKV-specific T-cells was associated with short clearance time. Our data thus demonstrate how the diversity and the exhaustion state of the T-cells can determine the clinical course of BKV infection.

10.
Transplantation ; 103(8): 1544-1555, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31033649

RESUMO

There is a clear medical need to change the current strategy of "one-size-fits-all" immunosuppression for controlling transplant rejection to precision medicine and targeted immune intervention. As T cells play a key role in both undesired graft rejection and protection, a better understanding of the fate and function of both alloreactive graft-deteriorating T cells and those protecting to infections is required. The T-cell receptor (TCR) is the individual identity card of each T cell clone and can help to follow single specificities. In this context, tracking of lymphocytes with certain specificity in blood and tissue in clinical follow up is of especial importance. After overcoming technical limitations of the past, novel molecular technologies opened new avenues of diagnostics. Using advantages of next generation sequencing, a method was established for T-cell tracing by detection of variable TCR region as identifiers of individual lymphocyte clones. The current review describes principles of laboratory and computational methods of TCR repertoire analysis, and gives an overview on applications for the basic understanding of transplant biology and immune monitoring. The review also delineates methodological pitfalls and challenges. With the outlook on prediction of antigens in immune-mediated processes including those of unknown causative pathogens, monitoring the fate and function of individual T cell clones, and the adoptive transfer of protective effector or regulatory T cells, this review highlights the current and future capability of TCR repertoire analysis.

11.
BMC Immunol ; 20(1): 11, 2019 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-31029086

RESUMO

BACKGROUND: Acute cellular rejection (ACR) is associated with complications after kidney transplantation, such as graft dysfunction and graft loss. Early risk assessment is therefore critical for the improvement of transplantation outcomes. In this work, we retrospectively analyzed a pre-transplant HLA antigen bead assay data set that was acquired by the e:KID consortium as part of a systems medicine approach. RESULTS: The data set included single antigen bead (SAB) reactivity profiles of 52 low-risk graft recipients (negative complement dependent cytotoxicity crossmatch, PRA < 30%) who showed detectable pre-transplant anti-HLA 1 antibodies. To assess whether the reactivity profiles provide a means for ACR risk assessment, we established a novel approach which differs from standard approaches in two aspects: the use of quantitative continuous data and the use of a multiparameter classification method. Remarkably, it achieved significant prediction of the 38 graft recipients who experienced ACR with a balanced accuracy of 82.7% (sensitivity = 76.5%, specificity = 88.9%). CONCLUSIONS: The resultant classifier achieved one of the highest prediction accuracies in the literature for pre-transplant risk assessment of ACR. Importantly, it can facilitate risk assessment in non-sensitized patients who lack donor-specific antibodies. As the classifier is based on continuous data and includes weak signals, our results emphasize that not only strong but also weak binding interactions of antibodies and HLA 1 antigens contain predictive information. TRIAL REGISTRATION: ClinicalTrials.gov NCT00724022 . Retrospectively registered July 2008.

12.
Methods Mol Biol ; 1926: 103-115, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30742266

RESUMO

Human pluripotent stem cells (hPSCs) have been well known for their ability to generate kidney cell types. We developed a protocol that utilizes a set of growth factors to give rise to kidney progenitors, which when differentiated further in a monolayer gives rise to podocyte precursors, mesangial cells, proximal and distal tubular epithelial cells, and collecting duct cells. This article describes in detail how to obtain each of these segment-specific kidney cell types from hPSCs. Once obtained as a homogenous population, these cells are invaluable for nephrotoxicity testing, for disease modeling, and in tissue engineering approaches such as 3D bioprinting and seeding on acellular matrices and scaffolds.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Rim/citologia , Organoides/citologia , Células-Tronco Pluripotentes/citologia , Técnicas de Cultura de Células/métodos , Diferenciação Celular/fisiologia , Humanos , Túbulos Renais/citologia
13.
Trends Mol Med ; 25(2): 149-163, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30711482

RESUMO

Intravascular infusion is the most popular route for therapeutic multipotent mesenchymal stromal/stem cell (MSC) delivery in hundreds of clinical trials. Meta-analysis has demonstrated that bone marrow MSC infusion is safe. It is not clear if this also applies to diverse new cell products derived from other sources, such as adipose and perinatal tissues. Different MSC products display varying levels of highly procoagulant tissue factor (TF) and may adversely trigger the instant blood-mediated inflammatory reaction (IBMIR). Suitable strategies for assessing and controlling hemocompatibility and optimized cell delivery are crucial for the development of safer and more effective MSC therapies.

14.
Front Immunol ; 10: 150, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30804944

RESUMO

CD4+CD25+FoxP3+ human regulatory TCELLS (TREG) are promising candidates for reshaping undesired immunity/inflammation by adoptive cell transfer, yet their application is strongly dependent on robust assays testing their functionality. Several studies along with first clinical data indicate TREG to be auspicious to use for future cell therapies, e.g., to induce tolerance after solid organ transplantation. To this end, TREG suppressive capacity has to be thoroughly evaluated prior to any therapeutic application. A 7 h-protocol for the assessment of TREG function by suppression of the early activation markers CD154 and CD69 on CD4+CD25- responder TCELLS (TRESP) upon polyclonal stimulation via αCD3/28-coated activating microbeads has previously been published. Even though this assay has since been applied by various groups, the protocol comes with a critical pitfall, which is yet not corrected by the journal of its original publication. Our results demonstrate that the observed decrease in activation marker frequency on TRESP is due to competition for αCD3/28-coated microbeads as opposed to a TREG-attributable effect and therefore the protocol cannot further be used as a diagnostic test to assess suppressive TREG function.

15.
Artigo em Inglês | MEDLINE | ID: mdl-30452217

RESUMO

The interaction of oxygen with Ni-Cr(100) alloy surfaces is studied using Scanning Tunneling Microscopy (STM) and Spectroscopy (STS) to observe the initial steps of oxidation and formation of the alloy-oxide interface. The progression of oxidation was observed for Ni(100) and Ni-Cr(100) thin films including Ni-8wt.%Cr(100) and Ni-12wt.%Cr(100), which were grown on MgO(100) in-situ. These surfaces were exposed to between one and 150 L O2 at 500 ºC, and additional annealing steps were performed at 500 ºC and 600 ºC. Each oxidation and annealing step was studied with STM and STS, and differential conductance maps delivered spatially resolved information on doping and bandgap distributions. Initial NiO nucleation and growth begins along the step edges of the Ni-Cr alloy accompanied by the formation of small oxide particles on the terraces. The incubation period known in oxidation of Ni(100) is absent on Ni-Cr alloy surfaces illustrating the significant changes in surface chemistry triggered by Cr alloying. Step edge faceting is initiated by step edge decoration of a NiO-Ni (78) coincidence lattice, which is expressed in moiré patterns in the STM images. Small patches of NiO are susceptible to reduction during annealing, but additional oxidation steps stabilize the NiO, which has a cube-on-cube epitaxial interface and a NiO-Ni (67) coincidence lattice grows into the terrace. NiO regions are interspersed with areas covered predominantly with a novel cross-type reconstruction, which is interpreted tentatively as a Cr-rich, phase-separated region. Statistical analysis of the geometric features of the surface oxide including step edge heights, and NiO wedge angles illustrate the layer-by-layer growth mode of NiO in this pre-Cabrera Mott regime, and the restructuring of the alloy-oxide interface during the oxidation process. This experimental approach has offered greater insight into the progression of oxide growth in Ni-Cr thin films and underscores the dramatic impact of alloying on oxidation process in the pre Cabrera-Mott regime.

16.
J Cachexia Sarcopenia Muscle ; 9(6): 1079-1092, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30334381

RESUMO

BACKGROUND: Most current cell-based regenerative therapies are based on the indirect induction of the affected tissues repair. Xenogeneic cell-based treatment with expanded human placenta stromal cells, predominantly from fetal origin (PLX-RAD cells), were shown to mitigate significantly acute radiation syndrome (ARS) following high dose irradiation in mice, with expedited regain of weight loss and haematopoietic function. The current mechanistic study explores the indirect effect of the secretome of PLX-RAD cells in the rescue of the irradiated mice. METHODS: The mitigation of the ARS was investigated following two intramuscularly (IM) injected 2 × 106 PLX-RAD cells, 1 and 5 days following 7.7 Gy irradiation. The mice survival rate and their blood or bone marrow (BM) cell counts were followed up and correlated with multiplex immunoassay of a panel of related human proteins of PLX-RAD derived secretome, as well as endogenous secretion of related mouse proteins. PLX-RAD secretome was also tested in vitro for its effect on the induction of the migration of BM progenitors. RESULTS: A 7.7 Gy whole body mice irradiation resulted in ~25% survival by 21 days. Treatment with two IM injections of 2 × 106 PLX-RAD cells on days 1 and 5 after irradiation mitigated highly significantly the subsequent lethal ARS, with survival rate increase to nearly 100% and fast regain of the initial weight loss (P < 0,0001). This was associated with a significant faster haematopoiesis recovery from day 9 onwards (P < 0.01). Nine out of the 65 human proteins tested were highly significantly elevated in the mouse circulation, peaking on days 6-9 after irradiation, relative to negligible levels in non-irradiated PLX-RAD injected mice (P < 0.01). The highly elevated proteins included human G-CSF, GRO, MCP-1, IL-6 and lL-8, reaching >500 pg/mL, while MCP-3, ENA, Eotaxin and fractalkine levels ranged between ~60-160pg/mL. The detected radiation-induced PLX-RAD secretome correlated well with the timing of the fast haematopoiesis regeneration. The radiation-induced PLX-RAD secretome seemed to reinforce the delayed high levels secretion of related mouse endogenous cytokines, including GCSF, KC, MCP-1 and IL-6. Additional supportive in vitro studies also confirmed the ability of cultured PLX-RAD secretome to induce accelerated migration of BM progenitors. CONCLUSIONS: A well-regulated and orchestrated secretion of major pro-regenerative BM supporting secretome in high dose irradiated mice, treated with xenogeneic IM injected PLX-RAD cells, can explain the observed mitigation of ARS. This seemed to coincide with faster haematopoiesis regeneration, regain of severe weight loss and the increased survival rate. The ARS-related stress signals activating the IM injected PLX-RAD cells for the remote secretion of the relevant human proteins deserve further investigation.

17.
Cell Mol Life Sci ; 2018 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-30310934

RESUMO

Human pluripotent stem cells (hPSCs) provide a source for the generation of defined kidney cells and renal organoids applicable in regenerative medicine, disease modeling, and drug screening. These applications require the provision of hPSC-derived renal cells by reproducible, scalable, and efficient methods. We established a chemically defined protocol by application of Activin A, BMP4, and Retinoic acid followed by GDNF, which steered hPSCs to the renal lineage and resulted in populations of SIX2+/CITED1+ metanephric mesenchyme- (MM) and of HOXB7+/GRHL2+ ureteric bud (UB)-like cells already by 6 days. Transcriptome analysis corroborated that the PSC-derived cell types at day 8 resemble their renal vesicle and ureteric epithelial counterpart in vivo, forming tubular and glomerular renal cells 6 days later. We demonstrate that starting from hPSCs, our in vitro protocol generates a pool of nephrogenic progenitors at the renal vesicle stage, which can be further directed into specialized nephronal cell types including mesangial-, proximal tubular-, distal tubular, collecting duct epithelial cells, and podocyte precursors after 14 days. This simple and rapid method to produce renal cells from a common precursor pool in 2D culture provides the basis for scaled-up production of tailored renal cell types, which are applicable for drug testing or cell-based regenerative therapies.

18.
Nat Med ; 2018 Oct 29.
Artigo em Inglês | MEDLINE | ID: mdl-30374197

RESUMO

The discovery of the highly efficient site-specific nuclease system CRISPR-Cas9 from Streptococcus pyogenes has galvanized the field of gene therapy1,2. The immunogenicity of Cas9 nuclease has been demonstrated in mice3,4. Preexisting immunity against therapeutic gene vectors or their cargo can decrease the efficacy of a potentially curative treatment and may pose significant safety issues3-6. S. pyogenes is a common cause for infectious diseases in humans, but it remains unclear whether it induces a T cell memory against the Cas9 nuclease7,8. Here, we show the presence of a preexisting ubiquitous effector T cell response directed toward the most widely used Cas9 homolog from S. pyogenes (SpCas9) within healthy humans. We characterize SpCas9-reactive T cells within the CD4/CD8 compartments for multi-effector potency, cytotoxicity, and lineage determination. In-depth analysis of SpCas9-reactive T cells reveals a high frequency of SpCas9-reactive regulatory T cells that can mitigate SpCas9-reactive effector T cell proliferation and function in vitro. Our results shed light on T cell-mediated immunity toward CRISPR-associated nucleases and offer a possible solution to overcome the problem of preexisting immunity.

19.
BMC Nephrol ; 19(1): 237, 2018 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-30231851

RESUMO

BACKGROUND: Conversion from calcineurin inhibitor (CNI) therapy to a mammalian target of rapamycin (mTOR) inhibitor following kidney transplantation may help to preserve graft function. Data are sparse, however, concerning the impact of conversion on posttransplant diabetes mellitus (PTDM) or the progression of pre-existing diabetes. METHODS: PTDM and other diabetes-related parameters were assessed post hoc in two large open-label multicenter trials. Kidney transplant recipients were randomized (i) at month 4.5 to switch to everolimus or remain on a standard cyclosporine (CsA)-based regimen (ZEUS, n = 300), or (ii) at month 3 to switch to everolimus, remain on standard CNI therapy or convert to everolimus with reduced-exposure CsA (HERAKLES, n = 497). RESULTS: There were no significant differences in the incidence of PTDM between treatment groups (log rank p = 0.97 [ZEUS], p = 0.90 [HERAKLES]). The mean change in random blood glucose from randomization to month 12 was also similar between treatment groups in both trials for patients with or without PTDM, and with or without pre-existing diabetes. The change in eGFR from randomization to month 12 showed a benefit for everolimus versus comparator groups in all subpopulations, but only reached significance in larger subgroups (no PTDM or no pre-existing diabetes). CONCLUSIONS: Within the restrictions of this post hoc analysis, including non-standardized diagnostic criteria and limited glycemia laboratory parameters, these data do not indicate any difference in the incidence or severity of PTDM with early conversion from a CsA-based regimen to everolimus, or in the progression of pre-existing diabetes. TRIAL REGISTRATION: clinicaltrials.gov , NCT00154310 (registered September 2005) and NCT00514514 (registered August 2007); EudraCT ( 2006-007021-32 and 2004-004346-40 ).

20.
J Cachexia Sarcopenia Muscle ; 9(5): 880-897, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30230266

RESUMO

BACKGROUND: No regenerative approach has thus far been shown to be effective in skeletal muscle injuries, despite their high frequency and associated functional deficits. We sought to address surgical trauma-related muscle injuries using local intraoperative application of allogeneic placenta-derived, mesenchymal-like adherent cells (PLX-PAD), using hip arthroplasty as a standardized injury model, because of the high regenerative and immunomodulatory potency of this cell type. METHODS: Our pilot phase I/IIa study was prospective, randomized, double blind, and placebo-controlled. Twenty patients undergoing hip arthroplasty via a direct lateral approach received an injection of 3.0 × 108 (300 M, n = 6) or 1.5 × 108 (150 M, n = 7) PLX-PAD or a placebo (n = 7) into the injured gluteus medius muscles. RESULTS: We did not observe any relevant PLX-PAD-related adverse events at the 2-year follow-up. Improved gluteus medius strength was noted as early as Week 6 in the treatment-groups. Surprisingly, until Week 26, the low-dose group outperformed the high-dose group and reached significantly improved strength compared with placebo [150 M vs. placebo: P = 0.007 (baseline adjusted; 95% confidence interval 7.6, 43.9); preoperative baseline values mean ± SE: placebo: 24.4 ± 6.7 Nm, 150 M: 27.3 ± 5.6 Nm], mirrored by an increase in muscle volume [150 M vs. placebo: P = 0.004 (baseline adjusted; 95% confidence interval 6.0, 30.0); preoperative baseline values GM volume: placebo: 211.9 ± 15.3 cm3 , 150 M: 237.4 ± 27.2 cm3 ]. Histology indicated accelerated healing after cell therapy. Biomarker studies revealed that low-dose treatment reduced the surgery-related immunological stress reaction more than high-dose treatment (exemplarily: CD16+ NK cells: Day 1 P = 0.06 vs. placebo, P = 0.07 vs. 150 M; CD4+ T-cells: Day 1 P = 0.04 vs. placebo, P = 0.08 vs. 150 M). Signs of late-onset immune reactivity after high-dose treatment corresponded to reduced functional improvement. CONCLUSIONS: Allogeneic PLX-PAD therapy improved strength and volume of injured skeletal muscle with a reasonable safety profile. Outcomes could be positively correlated with the modulation of early postoperative stress-related immunological reactions.

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