Lineage In Patients Research Articles

Multi-drug resistant Staphylococcus epidermidis from chronic wounds impair healing in human wound model.

Venous leg ulcers (VLUs) represent one of the most prevalent types of chronic wounds characterised by perturbed microbiome and biofilm-forming bacteria. As one of the most abundant skin-commensal, Staphylococcus epidermidis is known as beneficial for the host, however, some strains can form biofilms and hinder wound healing. In this study, S. epidermidis distribution in VLUs and associated resistome were analysed in ulcer tissue from patients. Virulence of S. epidermidis isolates from VLUs were evaluated by whole genome sequencing, antimicrobial susceptibility testing, in vitro biofilm and binding assays, and assessment of biofilm-forming capability and pro-inflammatory potential using human ex vivo wound model. We demonstrated that S. epidermidis isolates from VLUs inhibit re-epithelialization through biofilm-dependent induction of IL-1β, IL-8, and IL-6 which was in accordance with impaired healing outcomes observed in patients. High extracellular matrix binding ability of VLU isolates was associated with antimicrobial resistance and expression levels of the embp and sdrG, responsible for bacterial binding to fibrinogen and fibrin, respectively. Finally, we showed that S. epidermidis from VLUs demonstrate pathogenic features with ability to impair healing which underscores the emergence of treatment-resistant virulent lineages in patients with chronic ulcers.

Reprogramming of myeloid cells and their progenitors in patients with non-medullary thyroid carcinoma

Myeloid cells, crucial players in antitumoral defense, are affected by tumor-derived factors and treatment. The role of myeloid cells and their progenitors prior to tumor infiltration is poorly understood. Here we show single-cell transcriptomics and functional analyses of the myeloid cell lineage in patients with non-medullary thyroid carcinoma (TC) and multinodular goiter, before and after treatment with radioactive iodine compared to healthy controls. Integrative data analysis indicates that monocytes of TC patients have transcriptional upregulation of antigen presentation, reduced cytokine production capacity, and overproduction of reactive oxygen species. Interestingly, these cancer-related pathological changes are partially removed upon treatment. In bone marrow, TC patients tend to shift from myelopoiesis towards lymphopoiesis, reflected in transcriptional differences. Taken together, distinct transcriptional and functional changes in myeloid cells arise before their infiltration of the tumor and are already initiated in bone marrow, which suggests an active role in forming the tumor immune microenvironment.

The cell body space occupied by the nucleus during the cell differentiation in human lymphocytic, granulocytic and erythroid cell lineages.

The present nuclear and cell body diameter measurements demonstrated size differences of the approximate cell space estimate occupied by the cell nucleus during the cell differentiation in lymphocytic, granulocytic and erythroid cell lineages. These lineages were used as convenient models because all differentiation steps were easily identified and accessible in diagnostic peripheral blood or bone marrow smears of blood donors (BDs), patients suffering from chronic lymphocytic leukemia (CLL), patients with chronic myeloid leukemia (CML) and refractory anemia (RA) of the myelodysplastic syndrome (MDS). The cell space occupied by the nucleus was constant and did not change during the cell differentiation in the lymphocytic cell lineages of BDs and CLL patients despite the decreased cell size. In contrary, the cell space occupied by the nucleus markedly decreased in differentiating cells of granulocytic and erythroid lineages of patients suffering from CML. In the erythroid cell lineage in patients with RA of MDS the small reduction of the cell space occupied by the nucleus during the differentiation was not significant. The measurements also indicated that in progenitor cells of all studied cell lineages nuclei occupied more than 70 % of the cell space. Thus, the nucleus-cytoplasmic morphological and functional equilibrium appeared to be characteristic for each differentiation step and each specific cell lineage.

Molecular detection of Mycobacterium tuberculosis sensu stricto in the soil of Niger

Mycobacterium tuberculosis (MTB) complex is comprising of pathogenic mycobacteria responsible for human and animal tuberculosis, a major public health problem in Niger. Although infected individuals are paramount sources of contamination, nevertheless alternative, neglected sources may play some role in minority forms of the infection. Accordingly, we investigated the presence of Mycobacterium tuberculosis complex in soil samples in Niger. A total of 103 soil samples were collected in six different areas in Niger in October and November 2018 and April and May 2020 from residential areas of tuberculosis patients. Screening PCR targeting M. tuberculosis complex CRISPR-Csm4 and Xpert MTB/RIF Ultra assay were applied to detect the M. tuberculosis complex. M. tuberculosis DNA was positively detected in five of 103 (5/103; 4.8%) soil samples (Dosso: one sample, Zinder: one sample and Niamey: three samples) using the CRISPR-Csm4 system. CRISPR-Csm4 gene sequence identified four M. tuberculosis sensu stricto (may be lineages 1, 3 or 4) and one M. tuberculosis L2 lineage (Beijing). Moreover, the five positive samples were confirmed by Xpert MTB/RIF Ultra assay as rifampicin-susceptible M. tuberculosis complex strains. However, culture remained negative after 42 days. In this study, we announced for the first time the presence of M. tuberculosis sensu stricto in the soil of Niger. Moreover, these detected lineages were identical to the dominant M. tuberculosis lineages in patients. The presence of common lineages of M. tuberculosis between the soil and human highlight the risk of transmission from the soil to human.

Elfunat in complex treatment of patients with portal hypertension in the early post-hemorrhagic period

The paper deals with the results of the study of the effect of 2-ethyl-6-methyl-3-hydroxypyridine succinate (Elfunat) on erythroid lineage in patients with alcoholic liver cirrhosis and a history of gastroesophageal variceal bleeding. The study compared the impact on the central nervous system and blood values. The results of this study are important for the management of anemia and encephalopathy in this category of patients.

Population-Specific Patterns of Epigenetic Defects in the B Cell Lineage in Patients With Systemic Lupus Erythematosus.

To determine the stage of B cell development at which a systemic lupus erythematosus (SLE)-associated DNA methylation signature originates in African American (AA) and European American (EA) subjects, and to assess whether epigenetic defects in B cell development patterns could be predictive of SLE status in individual and mixed immune cell populations. B cells from AA patients (n = 31) and EA patients (n = 49) with or without SLE were sorted using fluorescence-activated cell sorting into 5 B cell subsets. DNA methylation, measured at ~460,000 CpG sites, was interrogated in each subset. Enrichment analysis of transcription factor interaction at SLE-associated methylation sites was performed. A random forests algorithm was used to identify an epigenetic signature of SLE in the B cell subsets, which was then validated in an independent cohort of AA and EA patients and healthy controls. Regression analysis across all B cell stages resulted in identification of 60 CpGs that reached genome-wide significance for SLE-associated methylation differences (P ≤ 1.07 × 10-7 ). Interrogation of ethnicity-specific CpGs associated with SLE revealed a hypomethylated pattern that was enriched for interferon (IFN)-regulated genes and binding of EBF1 in AA patients (each P < 0.001). AA patients with SLE could be distinguished from healthy controls when the predictive model developed with the transitional B cell subset was applied to other B cell subsets (mean receiver operating characteristic [ROC] area under the curve [AUC] 0.98), and when applied to CD19+ pan-B cells (mean ROC AUC 0.95) and CD4+ pan-T cells (mean ROC AUC 0.97) from the independent validation cohort. These results indicate that SLE-specific methylation patterns are ethnicity dependent. A pattern of epigenetic changes near IFN-regulated genes early in B cell development is a hallmark of SLE in AA female subjects. EBF1 binding sites are highly enriched for significant methylation changes, implying that this may be a potential regulator of SLE-associated epigenetic changes.

S889 GRANULOCYTES AND MONO‐NUCLEAR CELLS FROM PATIENTS WITH MYELOPROLIFERATIVE DISORDERS HARBOUR PERSISTENT EVIDENCE OF DNA DAMAGE AND TP53 PATHWAY ACTIVATION THAT CORRELATES WITH JAK‐STAT ACTIVATION

Background:The BCR‐ABL‐negative myeloproliferative neoplasms (MPN) are characterised by constitutive activation of the JAK‐STAT pathway in haematopoietic progenitor cells (HPC), typically resulting from somatic mutations in one of three genes; JAK2, MPL or CALR. It is known from murine models that activation of the JAK‐STAT pathways results in hyperproliferation of HPCs in the bone marrow niche, and it has recently been shown that this leads to exhaustion and premature aging in a percentage of HPCs. The impact of the activation of TP53 in the context of cells harbouring MPN‐driver mutations remains unclear however, and whether these ‘TP53‐high HPCs’ contribute to the mature cell populations in MPNs or are out‐competed by ‘TP53‐low’, HPC remains unknown.Aims:To determine the effect of JAK‐STAT activation on DNA damage and TP53 pathways in mature myeloid cells of patients with myeloproliferative disorders.Methods:Peripheral blood samples from patients with essential thrombocythaemia (ET, n = 10), polycythaemia vera (PV, N = 11) and myelofibrosis (MF, n = 7) as well as healthy controls (NC, n = 11) were fractioned by gradient separation and bead selection into granulocyte (PMN), mononuclear (MNC) and T‐cell (TC) fraction. Transcriptomic profiling was performed by expression array and validated by RNA‐seq in a subset of patients. Cell fractionation and purity was confirmed by computational decomposition and supervised differential expression, co‐expression and gene set enrichment analysis was then performed to identify disease specific pathway mis‐regulation.Results:Both PMN and MNC fractions displayed strong enrichment for hallmark TP53 pathway activation and DNA damage pathways. As expected, no TP53 pathway activation was seen in the T‐cell fraction, confirming that this is restricted to the myeloid compartment. Interestingly, TP53 pathway activation directly correlated with JAK‐STAT pathway activation, suggesting the p53 activation results directly from the effect of the MPN‐driver mutations.Given that TP53 is capable of activating a number of biological processes including cell cycle arrest, apoptosis and autophagy we looked at expression levels of a number of transcriptional targets of TP53. There was significantly increased expression of the cyclin‐dependent kinase inhibitor p21 (CDKN1A) a well‐known transcriptional target of p53, in the MNC fraction in all disease groups, and in the PMN fractions of PV and MF. Additionally, we noted upregulation of anti‐apoptotic BCL‐XL (BCL2L1) in a manner that mimicked CDKN1A upregulation.Summary/Conclusion:These data show that mature myeloid lineages in patients with MPNs harbour activation of DNA damage and TP53 pathways with transcriptional activation of down‐stream effectors in PMN and MNC compartments that correlate with JAK‐STAT activation. Typically, engagement of TP53 pathways serves to limit proliferation of mutation harbouring cells, however the upregulation of anti‐apoptotic genes may offer a potential mechanism for the persistent of these cells. These data suggest that MPN clones may develop mechanisms that permit escape from TP53 mediated death, and offers potential insight into novel therapeutic targets.

Altered Populations of Unconventional T Cell Lineages in Patients with Langerhans Cell Histiocytosis

Langerhans cell histiocytosis (LCH) lesions are defined by the presence of CD1a+/CD207+ myeloid cells, but many other immune cells are present including unconventional T cells, which have powerful immunoregulatory functions. Unconventional T cell lineages include mucosal-associated invariant T (MAIT) cells, type I natural killer T (NKT) cells and gamma-delta (γδ) T cells, which are associated with many inflammatory conditions, although their importance has not been studied in LCH. We characterized their phenotype and function in blood and lesions from patients with LCH, and identified a deficiency in MAIT cell frequency and abnormalities in the subset distributions of γδ T cells and NKT cells. Such abnormalities are associated with immune dysregulation in other disease settings and are therefore potentially important in LCH. Our study is the first to recognize alterations to MAIT cell proportions in patients with LCH. This finding along with other abnormalities identified amongst unconventional T cells could potentially influence the onset and progression of LCH, thereby highlighting potential targets for new immune based therapies.

Assessment of Erythroid and Granulocytic Hematopoietic Lineages in Patients with Non-Small-Cell Lung Carcinoma.

The toxic effects of combined cisplatin/docetaxel therapy cycles on erythroid and granulocytic hematopoietic lineages as well as their intercycle recovery were examined in patients with stage III-IV non-small-cell lung carcinoma. Responsiveness of the blood system to this therapy remained at a high level. Combined therapy pronouncedly activated the key elements of the erythroid and granulocytic hematopoietic lineages leading to accumulation of immature and mature myelokaryocytes in the bone marrow, enlargement of the medullary pool of mature neutrophils, and increase in the count of medullary erythroid and granulocytic precursor cells under conditions of their accelerated maturation.

Predictors and GVL Effects of UCB Chimerism after Haplo-Cord Transplant

Haplo cord transplant combines an umbilical cord blood unit (CBU) graft with a CD34 selected adult haplo-identical graft for the purpose of establishing long term CBU chimerism, which occurs in most but not all cases. Previously we showed that excessive haplo CD34 doses impair CBU engraftment (Liu et al, Blood 2011). Since then we have utilized a fixed haplo CD34 dose of 5x106 CD34 cells/kg. We also conducted a prospective study of CBU threshold dose de-escalation and established a minimum CBU dose of 1.2 x106 TNC/kg as the threshold for further study (van Besien ASH 2014, 1093).Here- using the patients from our prospective study- we sought to identify additional predictors of CBU engraftment as measured by percentage of CBU chimerism in CD33 and CD3 lineages in patients engrafted and in remission by day 56. We focused on d56 chimerism because durable patterns of chimerism are established by then.83 pts were enrolled, but 23 were excluded from analysis because of early death (6) relapse before day 60 (8), failure of both grafts with residual MDS (4), lost to follow up (1) and no chimerism measurements (3).61 pts were analyzed with median age 62 (18-72 years), 41 AML/MDS, 12 NHL, 5 ALL and 4 others. 24 had ASBMT high risk, 13 intermediate and 23 low risk disease. Conditioning was Flu Mel ATG (n=46), Flu Mel TBI 400 ATG (n=14) and Flu Mel TBI 600 ATG (n=1). All pts received GVHD prophylaxis with tacrolimus until d 180 and mycophenolate until d 30. The haplo dose was fixed at 5 x 106 CD34 cells/kg. Donors targeted by recipient donor specific antibodies (DSA) were avoided if possible. CBU units were selected based on HLA matching in sequential cohorts of decreasing threshold cell dose. Coh 1(n=12), minimum 2.0 x106 TNC/kg, coh 2 min (n=14) 1.5 x106 TNC/kg, coh 3 (n=25) min 1.0 x106 TNC/kg. After min cell dose was established, further enrollment occurred with a min dose of 1.2 x106 TNC/kg (coh 4 n=10). Median CBU TNC across all cohorts was 2.27 x107/kg (1.0-8.3). CBU were matched (HR HLA) at 3/8 (n=1), 4/8 (n=7), 5/8 (n=18), 6/8 (n=14) 7/8 (n=16) and 8/8 (n=5). DSA against CBU were present in 7. Haplo graft was 4/8 (n=42), 5/8 (n=13), 6/8 (n=3) and 7/8 (n=3). DSA against haplo were present in 6. Median follow up for survivors is 16 mo (5 -34).Cord graft characteristics, matching, enrollment cohort, nor presence of DSA were associated with d56 CBU chimerism. There was a strong inverse association between degree of matching of the haplo donor and d56 CBU (Fig 1). 54 pts with ≤ 5/8 haplo HLA match had 68% d56CD33 UCB chimerism vs 14% for the six pts with 6/8 or 7/8 HLA match (p=0.004). There was also a significant association between use of TBI and cord blood engraftment. 15 pts who received TBI had 79% d56CD33 UCB chimerism vs 50% for the 46 pts without TBI (p=0.05).Decreasing d56UCB chimerism was strongly associated with cumulative relapse risk (Fig 2A). It was not associated with TRM. The associations were even stronger if only considering AML/MDS (Fig 2B). Best discrimination was with 40% CD33 and 80% CD3 (Table 1).Table 1Relapse rate at one yearDay 56CD3 <80% (22)CD3>80% (39)p valueCD33<40% (19)CD33>40% (42)p valueAll pts n= 6147%21%0.00652%19%0.0008CD3 <80% (15)CD3>80% (26)p valueCD33<40% (13)CD33>40% (28)p valueAML/MDS n=4160%14%0.000358%16%0.00007Conclusions: The establishment of a durable cord blood graft is heavily influenced by HLA matching of the haplo-graft. 6/8 and 7/8 HLA-matched haplo-graft which occur by chance in about 10% of cases, should probably be avoided. More intensive conditioning with the addition of low dose TBI may further favor establishment of the cord blood graft. Cell dose or HLA matching of the cord blood graft has less effect on its long-term establishment.Increasing cord blood chimerism is associated with a much reduced risk of relapse despite- not shown here- very low rates of chronic GVHD). Low levels of cord blood chimerism are associated with very high rates of relapse. These data provide further support for the GVL effects of CBU grafts, particularly in AML/MDS. [Display omitted] [Display omitted] [Display omitted] Disclosuresvan Besien:Miltenyi Biotec: Research Funding.

Altered Phenotype of β-Cells and Other Pancreatic Cell Lineages in Patients With Diffuse Congenital Hyperinsulinism in Infancy Caused by Mutations in the ATP-Sensitive K-Channel.

Diffuse congenital hyperinsulinism in infancy (CHI-D) arises from mutations inactivating the KATP channel; however, the phenotype is difficult to explain from electrophysiology alone. Here we studied wider abnormalities in the β-cell and other pancreatic lineages. Islets were disorganized in CHI-D compared with controls. PAX4 and ARX expression was decreased. A tendency toward increased NKX2.2 expression was consistent with its detection in two-thirds of CHI-D δ-cell nuclei, similar to the fetal pancreas, and implied immature δ-cell function. CHI-D δ-cells also comprised 10% of cells displaying nucleomegaly. In CHI-D, increased proliferation was most elevated in duct (5- to 11-fold) and acinar (7- to 47-fold) lineages. Increased β-cell proliferation observed in some cases was offset by an increase in apoptosis; this is in keeping with no difference in INSULIN expression or surface area stained for insulin between CHI-D and control pancreas. However, nuclear localization of CDK6 and P27 was markedly enhanced in CHI-D β-cells compared with cytoplasmic localization in control cells. These combined data support normal β-cell mass in CHI-D, but with G1/S molecules positioned in favor of cell cycle progression. New molecular abnormalities in δ-cells and marked proliferative increases in other pancreatic lineages indicate CHI-D is not solely a β-cell disorder.

Ascertaining in vivo virulence of Mycobacterium tuberculosis lineages in patients in Mbeya, Tanzania.

We evaluated the relationship between the degree of immunodeficiency indicated by the number of circulating CD4+ T-cells and Mycobacterium tuberculosis lineages identified by spoligotyping and mycobacterial interspersed repetitive units-variable number of tandem repeats genotyping in human immunodeficiency virus (HIV) infected individuals with pulmonary tuberculosis from Mbeya, Tanzania. Of M. tuberculosis strains from 129 patients, respectively 55 (42.6%) and 37 (28.7%) belonged to Latin American Mediterranean and Delhi/Central-Asian lineages, while 37 (28.7%) patients were infected with other strains. There was no difference in the distribution of M. tuberculosis lineages among patients with early or advanced stages of HIV infection (P = 0.785), indicating that the virulence of strains from these lineages may not be substantially different in vivo.

Tracing the roots of cancer evolution.

By comparing the genomes of progenitor cells and mature cells of lymphoid and myeloid lineages in patients with chronic lymphocytic leukemia (CLL), Damm and colleagues confirmed that CLL originates from preleukemic CD34(+) progenitor cells and identified early CLL mutations that are associated with these progenitor cells. Moreover, they discovered that deregulation of B-cell receptor signaling may be one of the hallmarks of CLL, particularly in tumors with EGR2 mutations.

Both leukaemic and normal peripheral B lymphoid cells are highly sensitive to the selective pharmacological inhibition of prosurvival Bcl-2 with ABT-199

Overexpression of the prosurvival protein Bcl-2 marks many B-lymphoid malignancies and contributes to resistance to many commonly used chemotherapeutic agents. The first effective BH3 mimetic inhibitors of Bcl-2, ABT-737 and navitoclax, also target Bcl-xL, causing dose-limiting thrombocytopenia. This prompted the development of the Bcl-2-selective antagonist, ABT-199. Here we show that in lymphoid cells, ABT-199 specifically causes Bax/Bak-mediated apoptosis that is triggered principally by the initiator BH3-only protein Bim. As expected, malignant cells isolated from patients with chronic lymphocytic leukaemia are highly sensitive to ABT-199. However, we found that normal, untransformed mature B cells are also highly sensitive to ABT-199, both in vitro and in vivo. By contrast, the B-cell precursors are largely spared, as are cells of myeloid origin. These results pinpoint the probable impact of the pharmacological inhibition of Bcl-2 by ABT-199 on the normal mature haemopoietic cell lineages in patients, and have implications for monitoring during ABT-199 therapy as well as for the clinical utility of this very promising targeted agent.

Assessment of normal erythropoiesis by flow cytometry: important considerations for specimen preparation

The extension of quantitative flow cytometric studies to the erythroid lineage in patients with suspected myelodysplastic syndrome has prompted a reassessment of cell surface antigen expression during normal erythropoiesis. Erythropoiesis in normal and pathologic bone marrows was studied to determine the expected antigenic relationships of maturing erythroid cells. A total of 200 bone marrow specimens were evaluated by multidimensional flow cytometry (MDF). Samples were prepared using either NH4 Cl lysis or Ficoll density gradient separation. Normal erythroid development is described as a two-step process observable with the intensity relationships between CD235a, CD71, CD45, CD105, CD34, CD117, and CD36. The variability of these intensities (CV) was determined. A comparison of processing techniques determined lysis is the optimal analytic technique for the analysis of early-stage erythroid cells. Nucleic acid staining with DRAQ5 revealed that Ficoll allows for the analysis of reticulocytes and mature erythrocytes otherwise eliminated by lysis. These data demonstrate while lysis alters the light scatter characteristics of erythroid precursors, it did not alter quantitative antigen expression or nucleic acid content. The expected variability in antigen intensities is defined. These studies provide a basis for a comparison of erythroid development between normal individuals and those with erythroid dysplasia associated with myelodysplastic syndromes.

To the nucleolar bodies (nucleoli) in cells of the lymphocytic lineage in patients suffering from B – chronic lymphocytic leukemia

The present study was undertaken to provide more information on nucleoli in lymphocytes of B - chronic lymphocytic leukemia. The computer assisted nucleolar and cytoplasmic RNA image densitometry, reflecting the nucleolar and cytoplasmic RNA concentration at the single cell level, demonstrated a remarkable stability during the differentiation and maturation of B- lymphocytes. In contrast, as it was expected, the nucleolar diameter during the lymphocytic development markedly decreased. Thus the nucleolar RNA content of leukemic B-lymphocytes was apparently related to the nucleolar size. In both immature and mature lymphocytes, the cytostatic treatment increased the incidence of micronucleoli, which represent the "inactive" type of nucleoli. However, the decreased values of the nucleolar diameter were statistically significant only in mature lymphocytes of treated patients. On the other hand, despite such observation, it must be mentioned that "large active" and "ring shaped resting" nucleoli were still present in immature and mature lymphocytes after the cytostatic therapy and such cells might represent a potential pool of proliferating cells. As it is generally accepted "large active nucleoli" with multiple fibrillar centers are known to be characteristic for proliferating cells. "Ring shaped resting nucleoli" are present in sleeping cells, which may be stimulated to return to the cell cycle and to proliferate again. In addition, the nucleolar RNA distribution also indicated that Gumprecht ghosts mostly originated from mature lymphocytes. Increased ratio of the nucleolar to cytoplasmic RNA density in Gumprecht ghosts or apoptotic cells and apoptotic bodies of the lymphocytic origin was related to the decreased cytoplasmic RNA concentration. The increased nucleolar size together with the markedly decreased cytoplasmic RNA concentration characteristic for Gumprecht ghosts just reflected the spreading of lymphocytes during smear preparations. In apoptotic cells or bodies of the lymphocytic origin, the "frozen" nucleolar RNA concentration accompanied by a reduced RNA concentration in the cytoplasm exhibited a remarkable similarity to the apoptotic process induced in vitro by the cytostatic treatment. B-chronic lymphocytic leukemia; lymphocytes; nucleolar classes; size; nucleolar RNA image density -concentration.

Borrelia burgdorferistimulation of chemokine secretion by cells of monocyte lineage in patients with Lyme arthritis

IntroductionJoint fluid in patients with Lyme arthritis often contains high levels of CCL4 and CCL2, which are chemoattractants for monocytes and some T cells, and CXCL9 and CXCL10, which are chemoattractants for CD4+ and CD8+ T effector cells. These chemokines are produced primarily by cells of monocyte lineage in TH1-type immune responses. Our goal was to begin to learn how infection with Borrelia burgdorferi leads to the secretion of these chemokines, using patient cell samples. We hypothesized that B. burgdorferi stimulates chemokine secretion from monocytes/macrophages in multiple ways, thereby linking innate and adaptive immune responses.MethodsPeripheral blood mononuclear cells (PBMC) from 24 Lyme arthritis patients were stimulated with B. burgdorferi, interferon (IFN)-γ, or both, and the levels of CCL4, CCL2, CXCL9 and CXCL10 were measured in culture supernatants. CD14+ monocytes/macrophages from PBMC and synovial fluid mononuclear cells (SFMC) were stimulated in the same way, using available samples. CXCR3, the receptor for CXCL9 and CXCL10, and CCR5, the receptor for CCL4, were assessed on T cells from PBMC and SFMC.ResultsIn patients with Lyme arthritis, B. burgdorferi but not IFN-γ induced PBMC to secrete CCL4 and CCL2, and B. burgdorferi and IFN-γ each stimulated the production of CXCL9 and CXCL10. However, with the CD14+ cell fraction, B. burgdorferi alone stimulated the secretion of CCL4; B. burgdorferi and IFN-γ together induced CCL2 secretion, and IFN-γ alone stimulated the secretion of CXCL9 and CXCL10. The percentage of T cells expressing CXCR3 or CCR5 was significantly greater in SFMC than PBMC, confirming that TH1 effector cells were recruited to inflamed joints. However, when stimulated with B. burgdorferi or IFN-γ, SFMC and PBMC responded similarly.ConclusionsB. burgdorferi stimulates PBMC or CD14+ monocytes/macrophages directly to secrete CCL4, but spirochetal stimulation of other intermediate cells, which are present in PBMC, is required to induce CD14+ cells to secrete CCL2, CXCL9 and CXCL10. We conclude that B. burgdorferi stimulates monocytes/macrophages directly and indirectly to guide innate and adaptive immune responses in patients with Lyme arthritis.

Simultaneous Haploidentically Mismatched Stem Cell Transplantation from Multi-Donors (MDT) in Humans Offers Better Engraftment as Compared to Single Haploidentical Donor.

Introduction: stem-cell-transplantation (SCT) aims to combine tumor cytoreduction and host replacement with donor cells. For patients with no matched sibling available for allogeneic SCT (approximately 70% of patients) an alternative option is SCT from a relative carrying only one identical HLA haplotype (haplo-SCT), which may provide a donor for almost every patient at optimal timing. In the small number of multi-cord blood transplants reported, it was found that usually only one unit engrafted faster, while the others were rejected. We hypothesized that multi-donors may improve the engraftment following haplo-SCT. Patients and methods: Fifteen ultra high-risk patients (median age 20y, 9–52, mostly after 1–2 previous SCTs and 13 of them with refractory disease) were included. Conditioning regimen was fludarabine based. All grafts were T cell depleted.Results: All 14 evaluable patients engrafted but one of them later rejected the graft. The time for neutrophil engraftment (0.5 & 1.0x109/L) was fast, 11d (9–24) and 12d (10–28) respectively. Platelets engrafted even earlier (20 & 100x109/L), 10d (8–45) and 14d (12–32) respectively. Three patients demonstrate continuous bi-donor hematopoiesis (follow up 72, 128 and 168d) while 11 patients converted to single donor hematopoiesis within a median of 21d (14–39). Comparison of MDT patients to an earlier patient group receiving a similar protocol with a single donor (SDT) showed better granulocyte and platelet engraftment (p<0.001 for both, figures 1 and 2). Additionally, no primary graft failure occurred in patients undergoing MDT, while in SDT it occurred in 9/38 patients (p<0.001). One patient in-which leukemia persisted throughout the conditioning, leukemic cells disappeared upon the conversion to single donor chimerism. Despite major blood type differences, no hemolysis related side-effects were noted. Eight patients developed GVHD (mostly grade 1/2).Conclusion: MDT is safe and induces fast and durable engraftment of both myeloid and thrombocyte lineages in patients undergoing haplo-SCT. Further studies should follow this report. [Display omitted] [Display omitted]

Quantitative Analysis of Flow Cytometry Immunophenotyping Data for Aiding in the Diagnosis of Myelodysplastic Syndromes (MDS).

Recent studies have shown that flow cytometry immunophenotyping is a promising tool aiding in the diagnosis of myelodysplastic syndromes (MDS). However, the majority of these studies apply only qualitative pattern analysis in their interpretation of immunophenotypic data. The goal of this study was to analyze immunophenotyping results quantitatively, investigating the potential of this approach for providing additional information useful diagnosing MDS. Using flow cytometry immunophenotyping, we studied 56 bone marrow specimens from 13 patients with well-defined MDS by morphologic, clinical, and/or cytogenetic findings (5 RA, 3 RARS, 2 RAEB grade 1, 2 RAEB grade 2 and 1 secondary to chemotherapy), 15 cytopenic patients (controls, age-matched with MDS patients) with non-MDS/non-clonal hematologic disorders receiving marrow evaluation for other reason (ITP, fever of unknown origin, or lymphoma staging), 8 patients with AML transformed from MDS (t-AML), 6 patients with de novo AML, and 7 patients (14 specimens) with regenerating marrow after stem cell transplantation. These samples were analyzed qualitatively as reported in the literature as well as quantitatively for percentages of T-cells (CD3+), B-cells (CD20+), NK cells (CD3−/CD56+), granulocytes (moderate CD45 intensity and high side scatter characteristics), monocytes (CD14+/CD11c+), blasts (defined by dim CD45 and low side scatter, CD34+ or CD117+), erythroid precursors (CD71+/CD45−) and plasma cells (bright CD38), CD4/CD8 ratio, percentages of granulocyte subsets (CD10+, CD10−, CD36+/CD64+, CD36−/CD64+, CD11b−/CD16−, CD11b+/CD16− or CD11b+/CD16+ granulocytes per total granulocytes), percentage of CD56+ monocytes, and percentages of erythorid precursors subset (glycophorin A+ or glycophorin A- erythroid precursors per total erythroid precursors). In agreement with previous studies, qualitative analysis of these data demonstrated abnormal patterns of expression in myeloid and erythroid lineages in patients with MDS and t-AML. However, these patterns are also observed in age-matched controls and patients with de novo AML or with regenerating marrow. The quantitative analysis showed significantly increased T-cells and a significantly decreased granulocyte subset of CD11b+/CD16− in MDS patients when compared to age-matched controls (9.0 +/− 6.7% vs. 4.4 +/− 3.0 %, p = 0.023 and 28.1 +/− 14.9% vs. 44.5 +/−12.9%, p = 0.004, student t-test). There were also trends for increased NK cells and CD4:CD8 ratios and decreased total granulocytes in MDS patients as compared to the age-matched controls (p = 0.097, 0.094 and 0.059, respectively, student t-test). Other immunophenotypic parameters demonstrated no significant differences between these two groups. Furthermore, the changes observed in MDS patients were also seen in patients with t-AML. Patients with de novo AML or regenerating marrow post stem cell transplantation showed a quantitative immunophenotypic pattern between that of MDS patients and age-matched controls. These findings suggest that quantitative analysis of flow cytometry immunophenotyping data can aid in the diagnosis in MDS as well as the identification of AML arising from background MDS. The latter is clinically significant since these patients carry worse prognosis than those with de novo AML and may require novel therapies.

Somatic stem cell plasticity: current evidence and emerging concepts.

In the 19 th century, mammalian tissues were first described to be composed of cells, leading to the claim that cells originate exclusively from other cells (“omnis cellula a cellula”) formulated by Virchow and Schwann, respectively [1,2]. At the beginning of the 20 th century, the concept of tissue stem cells as the basis for tissue regeneration was introduced: analyzing the phylogeny of hematopoiesis in the bone marrow solely based on morphological observations, Pappenheim postulated the existence of an undifferentiated stem cell (“gemeinsame Stammzelle”) giving rise to the plethora of blood cells via an intermediate state of progenitor cells (Fig. 1, [3]). In the 1950s, several groups corroborated the existence of the hematopoietic stem cell in the bone marrow by showing hematopoietic recovery from transplanted bone marrow after irradiation damage [4–6]. Till and McCulloch later traced hematopoietic repopulation capacity to clonogenic cells establishing spleen colony-forming units [7]. Subsequently, the concept of tissue regeneration from a small population of resident tissue stem cells was generally accepted, was extended to nonhematopoietic tissues such as gut and skin [8], and still is our understanding of adult tissue regeneration today, enriched by an immense body of descriptive data. In parallel, the principle of directed cellular proliferation underlay the understanding of the early stages in embryogenesis and, together with the cellular movement, led to the discovery of morphogenesis via germ layers in the early embryo [9]. With emerging technologies, it was 33 and 3 years ago that stem cells with the capacity to differentiate into all tissues of the adult organism were functionally isolated from preimplantation embryos in mice and humans, respectively, and were called embryonic stem (ES) cells [10–13]. Although the concept of stem cells in embryogenesis and stem cells in adult tissue regeneration were initially pursued in conceptually separate approaches, they merged again with the successful cloning of a mammal from the nucleus of an adult tissue cell 4 years ago [14]. These experiments established that the nuclei of at least some adult cells were capable of being reprogrammed and spurred several groups to reevaluate the differentiation capacity of adult tissue stem cells, leading to a number of reports on somatic stem cell plasticity over the last 3 years. Here, we will review the current evidence for stem cell plasticity. Following the chronology of discoveries, we will start from the broadening developmental potential of bone marrow–derived stem cells leading to the differentiation capacities of stem cells from nonhematopoietic tissues. We will discuss some of the potential caveats to the current work, and finally will speculate about the potential underlying mechanisms of transdifferentiation.