Identification of critical hemodilution by artificial intelligence in bone marrow assessed for minimal residual disease analysis in acute myeloid leukemia: The Cinderella method.

Minimal residual disease (MRD) detection is a strong predictor for survival and relapse in acute myeloid leukemia (AML). MRD can be either determined by molecular assessment strategies or via multiparameter flow cytometry. The degree of bone marrow (BM) dilution with peripheral blood (PB) increases with aspiration volume causing consecutive underestimation of the residual AML blast amount. In order to prevent false-negative MRD results, we developed Cinderella, a simple automated method for one-tube simultaneous measurement of hemodilution in BM samples and MRD level. The explainable artificial intelligence (XAI) Cinderella was trained and validated with the digital raw data of a flow cytometric "8-color" AML-MRD antibody panel in 126 BM and 23 PB samples from 35 patients. Cinderella predicted PB dilution with high accordance compared to the results of the Holdrinet formula (Pearson's correlation coefficient r = 0.94, R2  = 0.89, p < 0.001). Unlike conventional neuronal networks Cinderella calculated the distributions of 12 different cell populations that were assigned to true hematopoietic counterparts as a human in the loop (HIL) approach. Besides characteristic BM cells such as myelocytes and myeloid progenitor cells the XAI identified discriminating populations, which were not specific for BM or PB (e.g., T cell/NK cell subpopulations and CD45 negative cells) and considered their frequency differences. Thus, Cinderella represents a HIL-XAI algorithm capable to calculate the degree of hemodilution in BM samples with an AML MRD immunophenotype panel. It is explicable, transparent, and paves a simple way to prevent false negative MRD reports.

Monitoring multiple myeloma in the peripheral blood based on cell-free DNA and circulating plasma cells.

With the advent of novel, highly effective therapies for multiple myeloma (MM), classical serologic monitoring appears insufficient for response assessment and prediction of relapse. Moreover, serologic studies in MM are hampered by interference of therapeutic antibodies. The detection of malignant plasma cell clones by next generation sequencing (NGS) or multiparameter flow cytometry (MFC) circumvents these difficulties and can be performed in the peripheral blood (pB) by targeting circulating cell-free DNA (cfDNA) or circulating plasma cells (CPCs), thus also avoiding an invasive sampling procedure. Here, we applied NGS of VJ light chain (LC) rearrangements in cfDNA and MFC of magnetically-enriched CD138-positive CPCs (me-MFC) to investigate disease burden in unselected MM patients. Sequencing was successful for 114/130 (87.7%) cfDNA samples and me-MFC results were analyzable for 196/205 (95.6%) samples. MM clones were detectable in 38.9% of samples taken at initial diagnosis or relapse (ID/RD), but only in 11.8% of samples taken during complete remission (CR). Circulating MM plasma cells were present in 83.3% of ID/RD samples and 9.9% of CR samples. Residual disease assessment by NGS or me-MFC in samples taken during very good partial remission or CR was 80% concordant. Notably, 4/4 (NGS) and 5/8 (me-MFC) positive CR samples were from patients with oligo- or non-secretory myeloma. The time to progression was shorter if there was evidence of residual myeloma in the pB. Together, our findings indicate that our two novel analytical approaches accurately indicate the course of MM and may be particularly valuable for monitoring patients with serologically non-trackable disease.

Expression of ß-1,4-galactosyltransferases during Aging in Caenorhabditis elegans.

BACKGROUND: Altered plasma activity of ß-1,4-galac-tosyl-transferases (B4GALTs) is a novel candidate biomarker of human aging. B4GALT1 is assumed to be largely responsible for this activity increase, but how it modulates the aging process is unclear at present. OBJECTIVES: To determine how expression of B4GALT1 and other B4GALT enzymes changes during aging of an experimentally tractable model organism, Caenorhabditis elegans. METHODS: Targeted analysis of mRNA levels of all 3 C. elegans B4GALT family members was performed by qPCR in wild-type and in long-lived daf-2 (insulin/IGF1-like receptor)-deficient or germline-deficient animals. RESULTS: bre-4 (B4GALT1/2/3/4) is the only B4GALT whose expression increases during aging in wild-type worms. In addition, bre-4 levels also rise during aging in long-lived daf-2-deficient worms, but not in animals that are long-lived due to the lack of germline stem cells. On the other hand, expression of sqv-3 (B4GALT7) and of W02B12.11 (B4GALT5/6) appears decreased or constant, respectively, in all backgrounds during aging. CONCLUSIONS: The age-dependent bre-4 mRNA increase in C. elegans parallels the age-dependent B4GALT activity increase in humans and is consistent with C. elegans being a suitable experimental organism to define potentially conserved roles of B4GALT1 during aging.

Antithetical NFATc1-Sox2 and p53-miR200 signaling networks govern pancreatic cancer cell plasticity.

In adaptation to oncogenic signals, pancreatic ductal adenocarcinoma (PDAC) cells undergo epithelial-mesenchymal transition (EMT), a process combining tumor cell dedifferentiation with acquisition of stemness features. However, the mechanisms linking oncogene-induced signaling pathways with EMT and stemness remain largely elusive. Here, we uncover the inflammation-induced transcription factor NFATc1 as a central regulator of pancreatic cancer cell plasticity. In particular, we show that NFATc1 drives EMT reprogramming and maintains pancreatic cancer cells in a stem cell-like state through Sox2-dependent transcription of EMT and stemness factors. Intriguingly, NFATc1-Sox2 complex-mediated PDAC dedifferentiation and progression is opposed by antithetical p53-miR200c signaling, and inactivation of the tumor suppressor pathway is essential for tumor dedifferentiation and dissemination both in genetically engineered mouse models (GEMM) and human PDAC. Based on these findings, we propose the existence of a hierarchical signaling network regulating PDAC cell plasticity and suggest that the molecular decision between epithelial cell preservation and conversion into a dedifferentiated cancer stem cell-like phenotype depends on opposing levels of p53 and NFATc1 signaling activities.

Comprehensive genetic diagnosis of acute myeloid leukemia by next-generation sequencing.

Differential induction therapy of all subtypes of acute myeloid leukemia other than acute promyelocytic leukemia is impeded by the long time required to complete complex and diverse cytogenetic and molecular genetic analyses for risk stratification or targeted treatment decisions. Here, we describe a reliable, rapid and sensitive diagnostic approach that combines karyotyping and mutational screening in a single, integrated, next-generation sequencing assay. Numerical karyotyping was performed by low coverage whole genome sequencing followed by copy number variation analysis using a novel algorithm based on in silico-generated reference karyotypes. Translocations and DNA variants were examined by targeted resequencing of fusion transcripts and mutational hotspot regions using commercially available kits and analysis pipelines. For the identification of FLT3 internal tandem duplications and KMT2A partial tandem duplications, we adapted previously described tools. In a validation cohort including 22 primary patients' samples, 9/9 numerically normal karyotypes were classified correctly and 30/31 (97%) copy number variations reported by classical cytogenetics and fluorescence in situ hybridization analysis were uncovered by our next-generation sequencing karyotyping approach. Predesigned fusion and mutation panels were validated exemplarily on leukemia cell lines and a subset of patients' samples and identified all expected genomic alterations. Finally, blinded analysis of eight additional patients' samples using our comprehensive assay accurately reproduced reference results. Therefore, calculated karyotyping by low coverage whole genome sequencing enables fast and reliable detection of numerical chromosomal changes and, in combination with panel-based fusion-and mutation screening, will greatly facilitate implementation of subtype-specific induction therapies in acute myeloid leukemia.

Correction to: Optimizing conditions for labeling of mesenchymal stromal cells (MSCs) with gold nanoparticles: a prerequisite for in vivo tracking of MSCs.

The authors apologized for the unfortunate error in figure during publication of the article and they also explained that some of the solid grey graphs in Fig. 5 are intentionally based on the same data. For 8 different surface makers (CD14, CD73, CD34, CD105, CD19, CD90, CD45, HA-DR) in accordance to the guidelines of the manufacturer a panel of 4 different isotype controls were used, corresponding to 4 different fluorescence channels.

Optimizing conditions for labeling of mesenchymal stromal cells (MSCs) with gold nanoparticles: a prerequisite for in vivo tracking of MSCs.

BACKGROUND: Mesenchymal stromal cells (MSCs) have an inherent migratory capacity towards tumor tissue in vivo. With the future objective to quantify the tumor homing efficacy of MSCs, as first step in this direction we investigated the use of inorganic nanoparticles (NPs), in particular ca. 4 nm-sized Au NPs, for MSC labeling. Time dependent uptake efficiencies of NPs at different exposure concentrations and times were determined via inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: The labeling efficiency of the MSCs was determined in terms of the amount of exocytosed NPs versus the amount of initially endocytosed NPs, demonstrating that at high concentrations the internalized Au NPs were exocytosed over time, leading to continuous exhaustion. While exposure to NPs did not significantly impair cell viability or expression of surface markers, even at high dose levels, MSCs were significantly affected in their proliferation and migration potential. These results demonstrate that proliferation or migration assays are more suitable to evaluate whether labeling of MSCs with certain amounts of NPs exerts distress on cells. However, despite optimized conditions the labeling efficiency varied considerably in MSC lots from different donors, indicating cell specific loading capacities for NPs. Finally, we determined the detection limits of Au NP-labeled MSCs within murine tissue employing ICP-MS and demonstrate the distribution and homing of NP labeled MSCs in vivo. CONCLUSION: Although large amounts of NPs improve contrast for imaging, duration and extend of labeling needs to be adjusted carefully to avoid functional deficits in MSCs. We established an optimized labeling strategy for human MSCs with Au NPs that preserves their migratory capacity in vivo.

Contact-dependent abrogation of bone marrow-derived plasmacytoid dendritic cell differentiation by murine mesenchymal stem cells.

Plasmacytoid dendritic cells (pDCs) are rare central regulators of antiviral immunity and unsurpassed producers of interferon-α (IFN-α). Despite their crucial role as a link between innate and adaptive immunity, little is known about the modulation of pDC differentiation by other bone marrow (BM) cells. In this study, we investigated the modulation of pDC differentiation in Flt-3 ligand (Flt3L)-supplemented BM cultures, using highly purified mesenchymal stem cells (MSCs) that were FACS-isolated from murine BM based on surface marker expression and used after in vitro expansion. Initial analysis revealed an almost complete inhibition of BM-derived pDC expansion in the presence of >2% MSC. This inhibition was cell contact-dependent and soluble factor-independent, as indicated by trans-well experiments. The abrogation of functional pDC development by MSCs was confirmed after TLR9 stimulation, revealing a complete, contact-dependent suppression of the IFN-a producing capacity of pDCs in Flt3L MSC BM co-cultures. MSC selectively inhibited pDC development in contrast to myeloid DC development, as indicated by the significantly increased numbers of myeloid DC in Flt3L-supplemented BM cultures. The absence of significant MSC-mediated inhibitory effects on myeloid DC differentiation was confirmed by additional experiments in GM-CSF/IL-4-supplemented BM cultures. In summary, we describe a novel contact-dependent immunomodulatory mechanism of MSC that targets the BM-derived expansion of functional pDCs.

A rational two-step approach to KRAS mutation testing in colorectal cancer using high resolution melting analysis and pyrosequencing.

BACKGROUND: KRAS mutation testing is mandatory in the management of metastatic colorectal cancer prior to treatment with anti-EGFR antibodies as patients whose tumors express mutant KRAS do not benefit from these agents. Although the U.S. Food and Drug Administration has recently approved two in-vitro diagnostics kits for determination of KRAS status, there is generally no consensus on the preferred method and new tests are continuously being developed. Most of these techniques focus on the hotspot mutations at codons 12 and 13 of the KRAS gene. METHODS: We describe a two-step approach to KRAS codon 12/13 mutation testing involving high resolution melting analysis (HRM) followed by pyrosequencing using the Therascreen KRAS Pyro kit (Qiagen) of only those samples that are not clearly identified as KRAS wildtype or mutant by HRM. First, we determined KRAS status in a panel of 61 colorectal cancer samples using both methods to compare technical performance and concordance of results. Subsequently, we evaluated practicability and costs of our concept in an independent set of 120 colorectal cancer samples in a routine diagnostic setting. RESULTS: HRM and pyrosequencing appeared to be equally sensitive, allowing for clear detection of mutant alleles at a mutant allele frequency ≥12.5 %. Pyrosequencing yielded more exploitable results due to lower input requirements and a lower rate of analysis failures. KRAS codon 12/13 status was called concordantly for 98.2 % (56/57) of all samples that could be successfully analysed by both methods and 100 % (19/19) of samples that were identified mutant by HRM. Reviewing the actual effort and expenses for KRAS mutation testing in our laboratory revealed, that the selective use of pyrosequencing for only those samples that could not be analysed by HRM increased the fraction of valid results from 87.5 % for HRM alone to 99.2 % (119/120) while allowing for a net reduction of operational costs of >75 % compared to pyrosequencing alone. CONCLUSIONS: Combination of HRM and pyrosequencing in a two-step diagnostic procedure constitutes a reliable and economic analysis platform for KRAS mutation testing in colorectal cancer in a clinical setting.

The inverse agonist DG172 triggers a PPARß/δ-independent myeloid lineage shift and promotes GM-CSF/IL-4-induced dendritic cell differentiation.

The stilbene derivative (Z)-2-(2-bromophenyl)-3-{[4-(1-methylpiperazine)amino]phenyl}acrylonitrile (DG172) was developed as a highly selective inhibitory peroxisome proliferator-activated receptor (PPAR)ß/δ ligand. Here, we describe a novel PPARß/δ-independent, yet highly specific, effect of DG172 on the differentiation of bone marrow cells (BMCs). DG172 strongly augmented granulocyte-macrophage-colony-stimulating factor (GM-CSF)-induced differentiation of primary BMCs from Ppard null mice into two specific populations, characterized as mature (CD11c(hi)MHCII(hi)) and immature (CD11c(hi)MHCII(lo)) dendritic cells (DCs). IL-4 synergized with DG172 to shift the differentiation from MHCII(lo) cells to mature DCs in vitro. The promotion of DC differentiation occurred at the expense of differentiation to granulocytic Gr1(+)Ly6B(+) cells. In agreement with these findings, transcriptome analyses showed a strong DG172-mediated repression of genes encoding neutrophilic markers in both differentiating wild-type and Ppard null cells, while macrophage/DC marker genes were up-regulated. DG172 also inhibited the expression of transcription factors driving granulocytic differentiation (Cebpe, Gfi1, and Klf5), and increased the levels of transcription factors promoting macrophage/DC differentiation (Irf4, Irf8, Spib, and Spic). DG172 exerted these effects only at an early stage of BMC differentiation induced by GM-CSF, did not affect macrophage-colony-stimulating factor-triggered differentiation to macrophages and had no detectable PPARß/δ-independent effect on other cell types tested. Structure-function analyses demonstrated that the 4-methylpiperazine moiety in DG172 is required for its effect on DC differentiation, but is dispensable for PPARß/δ binding. Based on these data we developed a new compound, (Z)-2-(4-chlorophenyl)-3-[4-(4-methylpiperazine-1-yl)phenyl]acrylonitrile (DG228), which enhances DC differentiation in the absence of significant PPARß/δ binding.

Mutations in SLC33A1 cause a lethal autosomal-recessive disorder with congenital cataracts, hearing loss, and low serum copper and ceruloplasmin.

Low copper and ceruloplasmin in serum are the diagnostic hallmarks for Menkes disease, Wilson disease, and aceruloplasminemia. We report on five patients from four unrelated families with these biochemical findings who presented with a lethal autosomal-recessive syndrome of congenital cataracts, hearing loss, and severe developmental delay. Cerebral MRI showed pronounced cerebellar hypoplasia and hypomyelination. Homozygosity mapping was performed and displayed a region of commonality among three families at chromosome 3q25. Deep sequencing and conventional sequencing disclosed homozygous or compound heterozygous mutations for all affected subjects in SLC33A1 encoding a highly conserved acetylCoA transporter (AT-1) required for acetylation of multiple gangliosides and glycoproteins. The mutations were found to cause reduced or absent AT-1 expression and abnormal intracellular localization of the protein. We also showed that AT-1 knockdown in HepG2 cells leads to reduced ceruloplasmin secretion, indicating that the low copper in serum is due to reduced ceruloplasmin levels and is not a sign of copper deficiency. The severity of the phenotype implies an essential role of AT-1 in proper posttranslational modification of numerous proteins, without which normal lens and brain development is interrupted. Furthermore, AT-1 defects are a new and important differential diagnosis in patients with low copper and ceruloplasmin in serum.

Prospectively defined murine mesenchymal stem cells inhibit Klebsiella pneumoniae-induced acute lung injury and improve pneumonia survival.

BACKGROUND: Numerous studies have described the immunosuppressive capacity of mesenchymal stem cells (MSC) but these studies use mixtures of heterogeneous progenitor cells for in vitro expansion. Recently, multipotent MSC have been prospectively identified in murine bone marrow (BM) on the basis of PDFGRa(+) SCA1(+) CD45(-) TER119(-) (PαS) expression but the immunomodulatory capacity of these MSC is unknown. METHODS: We isolated PαS MSC by high-purity FACS sorting of murine BM and after in vitro expansion we analyzed the in vivo immunomodulatory activity during acute pneumonia. PαS MSC (1 × 10(6)) were applied intratracheally 4 h after acute respiratory Klebsiella pneumoniae induced infection. RESULTS: PαS MSC treatment resulted in significantly reduced alveolitis and protein leakage in comparison to mock-treated controls. PαS MSC-treated mice exhibited significantly reduced alveolar TNF-α and IL-12p70 expression, while IL-10 expression was unaffected. Dissection of respiratory dendritic cell (DC) subsets by multiparameter flow cytometry revealed significantly reduced lung DC infiltration and significantly reduced CD86 costimulatory expression on lung CD103(+) DC in PαS MSC-treated mice. In the post-acute phase of pneumonia, PαS MSC-treated animals exhibited significantly reduced respiratory IL-17(+) CD4(+) T cells and IFN-γ(+) CD4(+) T cells. Moreover, PαS MSC treatment significantly improved overall pneumonia survival and did not increase bacterial load. CONCLUSION: In this study we demonstrated for the first time the feasibility and in vivo immunomodulatory capacity of prospectively defined MSC in pneumonia.

Immunosuppressive capabilities of mesenchymal stromal cells are maintained under hypoxic growth conditions and after gamma irradiation.

BACKGROUND AIMS: The discovery of regenerative and immunosuppressive capacities of mesenchymal stromal cells (MSCs) raises hope for patients with tissue-damaging or severe, treatment-refractory autoimmune disorders. We previously presented a method to expand human MSCs in a bioreactor under standardized Good Manufacturing Practice conditions. Now we characterized the impact of critical treatment conditions on MSCs with respect to immunosuppressive capabilities and proliferation. METHODS: MSC proliferation and survival after γ irradiation were determined by 5-carboxyfluorescein diacetate N-succinimidyl ester and annexinV/4',6-diamidino-2-phenylindole (DAPI) staining, respectively. T-cell proliferation assays were used to assess the effect of γ irradiation, passaging, cryopreservation, post-thaw equilibration time and hypoxia on T-cell suppressive capacities of MSCs. Quantitative polymerase chain reaction and ß-galactosidase staining served as tools to investigate differences between immunosuppressive and non-immunosuppressive MSCs. RESULTS: γ irradiation of MSCs abrogated their proliferation while vitality and T-cell inhibitory capacity were preserved. Passaging and long cryopreservation time decreased the T-cell suppressive function of MSCs, and postthaw equilibration time of 5 days restored this capability. Hypoxic culture markedly increased MSC proliferation without affecting their T-cell-suppressive capacity and phenotype. Furthermore, T-cell suppressive MSCs showed higher CXCL12 expression and less ß-galactosidase staining than non-suppressive MSCs. DISCUSSION: We demonstrate that γ irradiation is an effective strategy to abrogate MSC proliferation without impairing the cells' immunosuppressive function. Hypoxia significantly enhanced MSC expansion, allowing for transplantation of MSCs with low passage number. In summary, our optimized MSC expansion protocol successfully addressed the issues of safety and preservation of immunosuppressive MSC function after ex vivo expansion for therapeutic purposes.

Transcription factor IRF4 determines germinal center formation through follicular T-helper cell differentiation.

Follicular T-helper (T(FH)) cells cooperate with GL7(+)CD95(+) germinal center (GC) B cells to induce antibody maturation. Herein, we identify the transcription factor IRF4 as a T-cell intrinsic precondition for T(FH) cell differentiation and GC formation. After immunization with protein or infection with the protozoon Leishmania major, draining lymph nodes (LNs) of IFN-regulatory factor-4 (Irf4(-/-)) mice lacked GCs and GC B cells despite developing normal initial hyperplasia. GCs were also absent in Peyer's patches of naive Irf4(-/-) mice. Accordingly, CD4(+) T cells within the LNs and Peyer's patches failed to express the T(FH) key transcription factor B-cell lymphoma-6 and other T(FH)-related molecules. During chronic leishmaniasis, the draining Irf4(-/-) LNs disappeared because of massive cell death. Adoptive transfer of WT CD4(+) T cells or few L. major primed WT T(FH) cells reconstituted GC formation, GC B-cell differentiation, and LN cell survival. In support of a T-cell intrinsic IRF4 activity, Irf4(-/-) T(FH) cell differentiation was not rescued by close neighborhood to transferred WT T(FH) cells. Together with its known B lineage-specific roles during plasma cell maturation and class switch, our study places IRF4 in the center of antibody production toward T-cell-dependent antigens.

Low BCR-ABL expression levels in hematopoietic precursor cells enable persistence of chronic myeloid leukemia under imatinib.

BCR-ABL overexpression and stem cell quiescence supposedly contribute to the failure of imatinib mesylate (IM) to eradicate chronic myeloid leukemia (CML). However, BCR-ABL expression levels of persisting precursors and the impact of long-term IM therapy on the clearance of CML from primitive and mature bone marrow compartments are unclear. Here, we have shown that the number of BCR-ABL-positive precursors decreases significantly in all bone marrow compartments during major molecular remission (MMR). More importantly, we were able to demonstrate substantially lower BCR-ABL expression levels in persisting MMR colony-forming units (CFUs) compared with CML CFUs from diagnosis. Critically, lower BCR-ABL levels may indeed cause IM insensitivity, because primary murine bone marrow cells engineered to express low amounts of BCR-ABL were substantially less sensitive to IM than BCR-ABL-overexpressing cells. BCR-ABL overexpression in turn catalyzed the de novo development of point mutations to a greater extent than chemical mutagenesis. Thus, MMR is characterized by the persistence of CML clones with low BCR-ABL expression that may explain their insensitivity to IM and their low propensity to develop IM resistance through kinase point mutations. These findings may have implications for future treatment strategies of residual disease in CML.

Monitoring of acute myeloid leukemia patients after allogeneic stem cell transplantation employing semi-automated CD34+ donor cell chimerism analysis.

Determination of donor chimerism profiles in blood or bone marrow from patients with allogeneic stem cell transplantation (SCT) is useful for monitoring engraftment or predicting relapse, when specific molecular markers are lacking. CD34+ donor cell chimerism (DCC) analysis in peripheral blood samples from CD34+ acute myeloid leukemia (AML) and myleodysplastic syndrome (MDS) patients proved to be a highly sensitive diagnostic tool that is useful to detect imminent relapse significantly earlier compared to total white blood cell donor cell chimerism monitoring. However, flow-cytometric enrichment of CD34+ cells requires high efforts to human resources and equipment. We present a novel semi-automated CD34+ DCC analysis procedure-employing a magnetic cell-enrichment device, DNA extraction, and short tandem repeat profiling-without the need for flow-cytometric cell sorting. Monitoring 85 patients with AML and MDS over a period of 4 years 24 relapses were detected. Semi-automated peripheral blood CD34+ DCC was diminished below 80 % in all cases of systemic relapse. Significant decrease of the CD34+ DCC value was detected 29-42 days before overt cytological relapse. Our method provides a rapid and sensitive tool for monitoring AML and MDS patients after allogeneic SCT with regard to engraftment and early detection of relapse. Here, we propose a novel semi-automated procedure for CD34+ DCC analysis after allogeneic SCT that is simple, reliable, and therefore applicable in all hematologic laboratories.

Development of an explainable AI system using routine clinical parameters for rapid differentiation of inflammatory conditions.

Introduction: Inflammatory conditions in patients have various causes and require different treatments. Bacterial infections are treated with antibiotics, while these medications are ineffective against viral infections. Autoimmune diseases and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation, require immunosuppressive therapies such as glucocorticoids, which may be contraindicated in other inflammatory states. In this study, we employ a combination of straightforward blood tests to devise an explainable artificial intelligence (XAI) for distinguishing between bacterial infections, viral infections, and autoimmune diseases/graft-versus-host disease. Patients and methods: We analysed peripheral blood from 80 patients with inflammatory conditions and 38 controls. Complete blood count, CRP analysis, and a rapid flow cytometric test for myeloid activation markers CD169, CD64, and HLA-DR were utilized. A two-step XAI distinguished firstly with C5.0 rules pruned by ABC analysis between controls and inflammatory conditions and secondly between the types of inflammatory conditions with a new bivariate decision tree using the Simpson impurity function. Results: Inflammatory conditions were distinguished using an XAI, achieving an overall accuracy of 81.0% (95%CI 72 - 87%). Bacterial infection (N = 30), viral infection (N = 26), and autoimmune diseases/GVHD (N = 24) were differentiated with accuracies of 90.3%, 80.0%, and 79.0%, respectively. The most critical parameter for distinguishing between controls and inflammatory conditions was the expression of CD64 on neutrophils. Monocyte count and expression of CD169 were most crucial for the classification within the inflammatory conditions. Conclusion: Treatment decisions for inflammatory conditions can be effectively guided by XAI rules, straightforward to implement and based on promptly acquired blood parameters.

Good manufacturing practice-compliant animal-free expansion of human bone marrow derived mesenchymal stroma cells in a closed hollow-fiber-based bioreactor.

Mesenchymal stroma cells (MSC) are increasingly recognized for various applications of cell-based therapies such as regenerative medicine or immunomodulatory treatment strategies. Standardized large-scale expansions of MSC under good manufacturing practice (GMP)-compliant conditions avoiding animal derived components are mandatory for further evaluation of these novel therapeutic approaches in clinical trials. We applied a novel automated hollow fiber cell expansion system (CES) for in vitro expansion of human bone marrow derived MSC employing a GMP-compliant culture medium with human platelet lysate (HPL). Between 8 and 32 ml primary bone marrow aspirate were loaded into the hollow fiber CES and cultured for 15-27 days. 2-58 million MSC were harvested after primary culture. Further GMP-compliant cultivation of second passage MSC for 13 days led to further 10-20-fold enrichment. Viability, surface antigen expression, differentiation capacity and immunosuppressive function of MSC cultured in the hollow fiber CES were in line with standard criteria for MSC definition. We conclude that MSC can be enriched from primary bone marrow aspirate in a GMP-conform manner within a closed hollow fiber bioreactor and maintain their T lymphocyte inhibitory capacity. Standardized and reliable conditions for large scale MSC expansion pave the way for safe applications in humans in different therapeutic approaches.

α4 integrin levels on mobilized peripheral blood stem cells predict rapidity of engraftment in patients receiving autologous stem cell transplantation.

Rapidness of leukocyte engraftment in patients receiving peripheral blood stem cell transplantation is clinically important because the risk of fatal opportunistic infections increases with time to engraftment. Adhesion receptor molecules on hematopoietic stem cells (HSCs) have been shown to modulate homing and engraftment of HSCs. Therefore, we correlated expression levels of α4 (CD49d) and α6 (CD49f) integrins in the CD34(+) HSC compartment with time to engraftment. Leukapheresis products from 103 patients were retrospectively analyzed for CD34, CD38, CD3, CD49f, and CD49d surface molecules by multiparameter flow cytometry. High expression levels of α4 integrin, but not α6 integrin on CD34(+) cells, were associated with regular engraftment of leukocytes (days 8-19), whereas low surface expression correlated with delayed recovery (> 19 days; P < .0005). We show that α4 integrin expression levels on HSCs in leukapheresis products predict the engraftment capacity of mobilized peripheral blood stem cells in peripheral blood stem cell transplantation patients.